Novel proteins in enteroaggregative Escherichia coli (EAEC) useful for diagnosis and therapy of EAEC infections

ABSTRACT

Novel proteins and their corresponding nucleotide sequences in enteroaggregative  Escherichia coli  (EAEC) are provided. In particular, Aap and the five gene cluster (aat) of the AA probe region of the pAA plasmid of EAEC 042 have been identified, sequenced, and further characterized. The use of these novel proteins and their corresponding nucleotide sequences for diagnosis, therapy, and prevention of EAEC infections is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This non-provisional application is a Continuation of U.S. Ser. No.10/307,294, filed Dec. 2, 2002 (now allowed), which claims priorityunder 35 U.S.C. §119(e) to provisional applications U.S. Ser. Nos.60/334,425 and 60/398,775, filed Nov. 30, 2001 and Jul. 26, 2002,respectively, the contents of each of which are incorporated herein byreference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with U.S. Government support under NIADA grantNo. AI33096 awarded by the National Institutes of Health. The U.S.Government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to novel proteins and genes inenteroaggregative Escherichia coli (EAEC), and more particularly, to theuse of these proteins and their corresponding nucleotide sequences fordiagnosis, therapy, and prevention of EAEC infections.

BACKGROUND OF THE INVENTION

Enteroaggregative Escherichia coli (EAEC) is an emerging entericpathogen associated with sporadic, endemic, and epidemic diarrhealillnesses in individuals of all ages in both developing andindustrialized countries. (Nataro et al., Emerg Infect Dis 4:251-261(1998); Nataro et al., Clin Microbiol Rev. 11: 142-201 (1998); Okeke etal., Lancet Infect Dis 1:304-313 (2001)). The pathogenesis of EAECinfection includes strong adherence to the intestinal mucosa, mostlikely to both the small and the large intestines, followed by secretionof one or more enterotoxins that induce cytopathic effects in intestinalepithelial cells. (Nataro et al., Infect Immun 64:4761-4768 (1996);Eslava et al., Escherichia coli. Infect Immun 66:3155-3163 (1998);Czeczulin et al., Infect Immun 67:2692-2699 (1999)). Adherence of EAECto the intestinal mucosa is characterized by the presence of a thickaggregating biofilm, which may favor the persistence of this organism inthe human intestine. (Nataro et al., Clin Microbiol Rev. 11:142-201(1998); Tzipori et al., Infect Immun 60:5302-5306 (1992)). In addition,EAEC may induce intestinal inflammation, which can precipitate growthfailure even in the absence of diarrhea. (Steiner et al., J Infect Dis177:88-96 (1998)).

The defining feature of EAEC is its distinctive characteristicaggregative adherence (AA) pattern to HEp-2 cells in culture. (Nataro etal., Pediatr Infect Dis J 6:829-831 (1987)). In particular, EAEC adhereto the surface of HEp-2 cells, to the glass substratum, and to eachother in a distinctive stacked-brick formation. Aggregating adherence toHEp-2 cells in well-characterized strains requires the expression of oneor more members of the plasmid-borne Aggregative Adherence Fimbriae(AAF) family. (Nataro et al., Infect Immun 60:2297-2304 (1992);Czeczulin et al., Infect Immun 65:4135-4145 (1997)). Two of members ofthe AAF family, AAF/I and AAF/II, have been characterized at the geneticlevel and each is encoded on a large plasmid (designated pAA). (Savarinoet al., J Bacteriol 176:4949-57 (1994); Elias et al., J Bacteriol181:1779-85 (1999)). It has been shown that the human pathogenic strain042 requires AAF/II fimbrial antigen for adherence of the bacterium tothe colonic mucosa, thereby suggesting that this adhesin is a virulencefactor for human infection. (Czeczulin et al., Infect Immun 65:4135-4145(1997)).

It has been shown that the majority of EAEC strains lack AAF/I andAAF/II. (Czeczulin et al., Infect Immun 67:2692-2699 (1999)).Nevertheless, most EAEC strains carry the ca. 100 kb pAA plasmid,recognized by the presence of several conserved loci. The most prominentamong these loci is a transcriptional activator of the AraC classdesignated AggR, which is required for expression of both AAF/I andAAF/II. AggR is also present in a large percentage of EAEC strains thatdo not express any identified AAF. (Nataro et al., J Bacteriol176:4691-4699 (1994)). Recently, a novel AggR-dependent gene lyingimmediately upstream of AggR in EAEC 042 has been identified andcharacterized. (Sheikh et al., J Clin Invest (in press) (2002)). ThisAggR-dependent gene encodes a secreted 10.2 kD protein, designated Aap,that appears to coat the bacterial surface and promote dispersion ofEAEC on the intestinal mucosa. Aap has alternatively been designateddispersin. It has been shown that Aap mutants form larger aggregates,fewer individual bacteria, and aggregate more intensely than the wildtype. In addition, Aap partially counteracts AAF-mediated aggregationand may play a fundamental role in EAEC pathogenesis. Moreover, the datasuggest that Aap binds non-covalently to the bacterial cell surface.However, the mechanism by which Aap is translocated across the outermembrane is as yet unknown.

Another prominent locus is the binding site of a DNA probe, CVD432, onthe plasmid. The CVD432 probe was developed to simplify theidentification of EAEC and has been used as the AA probe. (Baudry etal., J Infect Dis 161:1249-1251 (1990)). In its original evaluation, theprobe was found to be 89% sensitive and 99% specific for EAEC. Thenucleotide sequence of the AA probe represented a cryptic open readingframe (ORF) located adjacent to the plasmid replicon. (Nataro et al.,Infect Immun 64:4761-4768 (1996)). Further sequence analysis of the AAprobe region revealed five ORFs. The predicted protein of one of theORFs was similar with the ATP-binding cassette (ABC) domain of the ABCtransporter, suggesting that the gene cluster is involved in thetransport of an unidentified molecule of EAEC. Furthermore, this genecluster appears to be associated with the translocation of Aap, and theformation of a new transporting system of the virulence factor of EAEC.The five gene cluster was designated aat.

EAEC infections are medically important in populations around the world.For example, in developing countries, children are often infected withEAEC and can develop prolonged diarrhea, often with significantmalnutrition. In industrialized countries, EAEC is an emerging cause oftraveler's diarrhea. An effective vaccine to protect againstenterotoxigenic E. coli would protect against at least one-half oftraveler's diarrhea cases. However, in the past, there has been noeffective treatment or prevention of EAEC-induced illnesses.

It is therefore desirable to identify and characterize Aap and the fivegene cluster (aat) of the AA probe region of the pAA plasmid of EAEC 042and to use these proteins and their corresponding nucleotide sequencesfor diagnosis, therapy, and prevention of EAEC infections.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to identify andcharacterize Aap, AatP, AatA, AatB, AatC, and AatD, and fragments ofthese proteins, for diagnosis, therapy, and prevention of EAECinfections. It is also an object of this invention to identify andcharacterize aap, aatP, aatA, aatB, aatC, and aatD (the aat gene clusteror the aat cluster), and fragments of these genes, for diagnosis,therapy, and prevention of EAEC infections.

One embodiment of the present invention relates to an immunogeniccomposition that includes a recombinant product of Aap, AatP, AatA,AatB, AatC, or AatD and a carrier. Various aspects of this embodimentrelate to compositions in which the recombinant product of Aap, AatP,AatA, AatB, AatC, or AatD is a fragment of Aap, AatP, AatA, AatB, AatC,or AatD, respectively, full-length Aap, AatP, AatA, AatB, AatC, or AatD,respectively, or a product that is at least 95% homologous to Aap, AatP,AatA, AatB, AatC, or AatD respectively. The recombinant product of Aap,AatP, AatA, AatB, AatC, or AatD can comprise Aap, AatP, AatA, AatB,AatC, or AatD itself, respectively.

Another embodiment of the present invention relates to an isolatednucleotide sequence comprising aap, aatP, aatA, aatB, aatC, or aatD or afunctional fragment thereof.

Yet another embodiment of the present invention relates to a purifiedpolypeptide sequence encoding Aap, AatP, AatA, AatB, AatC, or AatD or afunctional equivalent of Aap, AatP, AatA, AatB, AatC, or AatD.

A further embodiment of the present invention relates to methods ofusing the gene encoding for aap, aatP, aatA, aatB, aatC, or aatD,products, and fragments thereof. One particular embodiment disclosedherein teaches a method of generating an immune response that includesproviding an immunogenic composition that includes Aap, AatP, AatA,AatB, AatC, or AatD, a functional fragment thereof, or a product thereofto a subject and contacting the subject with the immunogeniccomposition, which generates an immune response in the subject.

Another embodiment of the present invention relates to a method ofproducing a polypeptide product from a polynucleotide encoding aap,aatP, aatA, aatB, aatC, or aatD, or functional fragment thereof, thatincludes providing aap, aatP, aatA, aatB, aatC, or aatD in an expressionvector, introducing the expression vector into a host cell such that arecombinant host cell is produced, and subjecting the recombinant hostcell to conditions such that a protein from aap, aatP, aatA, aatB, aatC,or aatD is expressed.

An additional embodiment of the present invention relates to cellscomprising recombinant aap, aatP, aatA, aatB, aatC, or aatD, orfragments thereof, and expression vectors comprising aap, aatP, aatA,aatB, aatC, or aatD, or fragments thereof.

Yet another embodiment of the present invention relates to antibodiesand antibody fragments that bind to and recognize Aap, AatP, AatA, AatB,AatC, or AatD, or fragments of Aap, AatP, AatA, AatB, AatC, or AatD.

A further embodiment of the present invention relates to the diagnosisof diseases caused by enteroaggregative E. coli (EAEC). The diagnosis ofdiseases caused by EAEC can be performed by using antibodies orfragments of antibodies that bind to and recognize Aap, AatP, AatA,AatB, AatC, or AatD, or fragments of Aap, AatP, AatA, AatB, AatC, orAatD. An alternative embodiment of the present invention relating to thediagnosis of diseases caused by EAEC relates to the use ofpolynucleotides that are complementary to aap, aatP, aatA, aatB, aatC,or aatD, or portions of aap, aatP, aatA, aatB, aatC, or aatD to diagnosethe disease caused by EAEC.

Additional embodiments of the present invention encompass kits thatutilize antibodies or fragments of antibodies that bind to and recognizeAap, AatP, AatA, AatB, AatC, or AatD or fragments of Aap, AatP, AatA,AatB, AatC, or AatD, kits that utilize antibodies or other proteins(i.e., Aap, AatP, AatA, AatB, AatC, or AatD), and kits that utilizepolynucleotides that bind to and recognize aap, aatP, aatA, aatB, aatC,or aatD, or fragments of aap, aatP, aatA, aatB, aatC, or aatD. Thesekits can be used for the diagnosis of disease caused by EAEC and fordiagnosis of EAEC infections.

It is a feature of the present invention that isolated and purifiedpolynucleotide molecules encoding the Aap protein are capable ofhybridizing under moderate to stringent conditions to an oligonucleotideof 15 or more contiguous nucleotides of SEQ ID NO: 1 or itscomplementary strand.

It is another feature of the present invention that isolated andpurified polynucleotide molecules encoding the AatP protein are capableof hybridizing under moderate to stringent conditions to anoligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO: 9 orits complementary strand.

It is a yet another feature of the present invention that isolated andpurified polynucleotide molecules encoding the AatA protein are capableof hybridizing under moderate to stringent conditions to anoligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO: 10 orits complementary strand.

It is a further feature of the present invention that isolated andpurified polynucleotide molecules encoding the AatB protein are capableof hybridizing under moderate to stringent conditions to anoligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO: 11 orits complementary strand.

It is also a feature of the present invention that isolated and purifiedpolynucleotide molecules encoding the AatC protein are capable ofhybridizing under moderate to stringent conditions to an oligonucleotideof 15 or more contiguous nucleotides of SEQ ID NO: 12 or itscomplementary strand.

It is a further feature of the present invention that isolated andpurified polynucleotide molecules encoding the AatD protein are capableof hybridizing under moderate to stringent conditions to anoligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO: 13 orits complementary strand.

The foregoing and other objects, features, and advantages of theinvention will appear more fully hereinafter from a consideration of thedetailed description that follows, in conjunction with the accompanyingsheets of figures. It is to be expressly understood, however, that thedrawings are for illustrative purposes and are not to be construed asdefining the limits of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages of this invention will be apparent upon consideration ofthe following detailed disclosure of the invention, especially whentaken in conjunction with the accompanying drawings wherein:

FIG. 1 illustrates the nucleotide sequence of aap (SEQ ID NO:1);

FIG. 2 illustrates the amino acid sequence of Aap (SEQ ID NO:2);

FIG. 3 is a map of 7 kb fragment of the pAA plasmid of EAEC 042 showingthe location of the aat gene cluster;

FIG. 4A is a photograph of the SDS-PAGE of culture media precipitatedwith trichloroacetic acid of wild type and mutant aat;

FIG. 4B is a photograph of the SDS-PAGE of the supernatant of theculture with 0.1% Triton X-100 of wild type and mutant aat;

FIG. 5 is a photograph the Western Immunoblot of 042 and 042aatA insupernatant and cell pellet;

FIG. 6A is a photograph of a Western Immunoblot of secreted Aat in thesupernatant and cell pellet, and illustrates different levels ofexpression in wild type, 042aatC and 042aatD, but not in 042pet;

FIG. 6B is a photograph of a western immunoblot of levels of Aat wildtype, 042aatC, 042aatD and 042pet in periplasm;

FIG. 7A is a photograph of a Western Immunoblot of whole cells and theouter membrane of 042aatA(pJNW);

FIG. 7B is a photograph of a Western Immunoblot analysis for Aap in thesupernatant of 042, 042aatA, and 042aatA (pJNW);

FIG. 8 is a photograph of a Western Immunoblot illustrating that thecomplement 042aatC(pJNW) and 042aatD(pJNW) restores Aap secretion;

FIG. 9 illustrates the nucleotide sequence of the 7 kb fragment of pAAof EAEC 042 (SEQ ID NO: 3);

FIG. 10 illustrates the amino acid sequence of AatP (SEQ ID NO: 4).

FIG. 11 illustrates the amino acid sequence of AatA (SEQ ID NO: 5).

FIG. 12 illustrates the amino acid sequence of AatB (SEQ ID NO: 6).

FIG. 13 illustrates the amino acid sequence of AatC (SEQ ID NO: 7).

FIG. 14 illustrates the amino acid sequence of AatD (SEQ ID NO: 8).

FIG. 15 illustrates the nucleotide sequence of aatP (SEQ ID NO: 9).

FIG. 16 illustrates the nucleotide sequence of aatA (SEQ ID NO: 10).

FIG. 17 illustrates the nucleotide sequence of aatB (SEQ ID NO: 11).

FIG. 18 illustrates the nucleotide sequence of aatC (SEQ ID NO: 12).

FIG. 19 illustrates the nucleotide sequence of aatD (SEQ ID NO: 13).

DETAILED DESCRIPTION OF THE INVENTION

This invention covers Aap (for Anti-aggregation protein, also calleddispersin), AatP, AatA, AatB, AatC, and AatD which are all proteinsfound in enteroaggregative E. coli (EAEC). This invention also coversthe genes for these proteins (aap, aatP, aatA, aatB, aatC, and aatD) andfragments of the genes and proteins, and use of these proteins, genes,and fragments thereof for diagnosis and therapy of EAEC infections. Thefollowing genes are also called the aat gene cluster: aatP, aatA, aatB,aatC, and aatD.

Aap is secreted from enteroaggregative E. coli (EAEC) and modulates thestrong aggregative phenotype of EAEC on the intestinal mucosa. Thesecretion of Aap does not negatively affect EAEC fimbrial expression orfimbrial biogenesis. Aap is a 116 amino acid protein, has a molecularweight of about 10.2 kilodaltons (kDa) as determined by SDS-PAGE, a pIof approximately 9.25, and an amino acid sequence illustrated in FIG. 2(SEQ ID NO: 2). SIGNALP analysis strongly predicts a signal sequencewith cleavage after position 21. An N-terminal amino acid sequence ofAap from bacterial culture supernatants confirms this cleavage.

aap is 348 base-pairs (bp) in length and has the polynucleotide sequenceillustrated in FIG. 1 (SEQ ID NO: 1). aap is located on the pAA plasmid(ca. 100 kp) of prototype EAEC strain 042 (EAEC 042) upstream of theaggR gene which is located downstream of the fimbrial subunit (aafA).Sequencing of the pAA plasmid DNA upstream of aggR revealed an openreading frame 843 nucleotides upstream from the aggR start codon. Thisopen reading frame was 348 nucleotides in length and encoded a predictedprotein product of 116 amino acids. Analysis with SIGNALP (a web-basedalgorithm which predicts the site of signal sequence processing)strongly predicted a signal sequence with cleavage after position 21.N-terminal amino acid sequence of Aap from bacterial culturesupernatants confirmed the cleavage signal sequence and the sequenceindicated in FIG. 2.

The pAA plasmid not only contains putative virulence factors AAF, Aap(dispersin), Pet, EAST1, and AggR but also contains sequences homologousto an empirically derived probe sensitive to EAEC strains (the “AAprobe”). (Baudrey et al., J Infect Dis 161:1249-1251 (1990)). The regioncomprising the AA probe (i.e., the insert of plasmid pCVD432) has beenshown to be associated with pathogenic EAEC strains and to correlatewith the presence of plausible virulence factors. (Okeke et al., JInfect Dis 81:252-60 (2000); Cohen, M. and Nataro, J. P., unpublished).Although the 765 bp sequence of the probe itself has been previouslyreported (Schmidt et al., J Clin Microbiol 33:701-5 (1995)), it wasnoted that the sequence did not contain stop codons in one potentialreading frame. A 7 kb sequence of the pAA2 plasmid was determined byshotgun sequencing of a pBluescript library (see FIG. 9 (SEQ ID NO: 3)).Discrepancies were resolved by directed sequencing of pBluescript clonescomprising the desired region.

Analysis of the DNA sequence of this region of the probe reveal acluster of five open reading frames (ORFs) in the same rightwardorientation and very closely spaced. (See FIG. 3). This cluster of ORF'sis known as the aat gene cluster or aat cluster because two of genes inthis cluster exhibit significant homology to bacterial ABC transporterproteins thus is short for enteroaggregative ABC transporter. The aatgene cluster features very low G+C ratios, ranging from 29.6 to 34.3.The aat gene cluster contains aatP, aatA, aatB, aatC, and aatD. Flankingthe aat gene cluster at distances of over 500 bp on each side areremnants of IS sequences.

The nucleotide sequence of aatP is shown in FIG. 15 (SEQ ID NO: 9). Theamino acid sequence for AatP is shown in (SEQ ID NO: 4). AatP is apredicted protein of 377 amino acids (42.7 kDa) and runs from nucleotide1425 to 2555 of the 7 kb fragment from pAA as shown in FIG. 9. AatP is20% identical at the amino acid level over its entire length to permeasecomponents of ABC-type transport systems that are involved inlipoprotein release. The protein has five transmembrane regions and isbe believed to be an inner membrane protein, based on computer analysis.

The nucleotide sequence of aatA is shown in FIG. 16 (SEQ ID NO: 10). Theamino acid sequence for AatA is shown in FIG. 11 (SEQ ID NO: 5). AatA isa predicted protein of 412 amino acids (48.5 kDa) and runs fromnucleotide 2552 to 3790 of the 7 kb fragment from pAA as shown in FIG. 9(SEQ ID NO: 3). SignalP analysis strongly predicts a signal sequencewith cleavage after position 23. The predicted mature protein of 391amino acids is 45.9 kDa in size. Neither the nucleotide sequence nor thededuced amino acid sequence of aatA display's significant identity toany other known gene or protein. AapA has a coiled coil region and isbelieved to be an outer membrane or periplasmic protein.

The nucleotide sequence of aatB is shown in FIG. 17 (SEQ ID NO: 11). Theamino acid sequence for AatB is shown in FIG. 12 (SEQ ID NO: 6). AatB isa predicted protein of 274 amino acids (31.1 kDa) and runs fromnucleotide 3687 to 4508 of the 7 kb fragment from pAA as shown in FIG. 9(SEQ ID NO: 3). AatB does not have any significant homology to any knownprotein sequences in the GenBank library. The protein has atransmembrane region in the N-terminus and is believed to be an innermembrane protein.

The nucleotide sequence of aatC is shown in FIG. 18 (SEQ ID NO: 12). Theamino acid sequence for AatC is shown in FIG. 13 (SEQ ID NO: 7). AatC isa predicted protein of 210 amino acids (23.3 kDa) and runs fromnucleotide 4501 to 5130 of the 7 kb fragment from pAA as shown in FIG. 9(SEQ ID NO: 3). AatC is 45% identical at the amino acid level over itsentire length to an ABC transporter ATP-binding protein. ABC domains ofE. coli have four short motifs that are invariably conserved: Walker A,Walker B, ABC signature, and the histidine motif (Linton 1998). Allthese motifs were conserved in AatC.

The nucleotide sequence of aatD is shown in FIG. 19 (SEQ ID NO: 13). Theamino acid sequence for AatD is shown is FIG. 14 (SEQ ID NO: 8). AatD isa predicted protein of 405 amino acids (47.2 kDa) and runs fromnucleotide 5142 to 6356 of the 7 kb fragment from pAA as shown in FIG. 9(SEQ ID NO: 3). AatD is 29% identical at the amino acid level over 34%of its length to NADH dehydrogenase subunit 2. AatD has fivetransmembrane regions in the N-terminal and is believed to be an innermembrane protein.

For each of the above discussed genes, additional polynucleotidemolecules that encode the proteins discussed above include thosepolynucleotide sequences resulting in minor genetic polymorphisms,differences between strains, degenerate variants, and encoding forproteins that contain amino acid substitutions, additions, and/ordeletions.

Nucleotide sequences encoding aap and/or the aat gene cluster can beused to identify polynucleotide molecules encoding other proteins withbiological functions similar to that of Aap and/or the proteins of theaat gene cluster by screening a cDNA library or DNA library from otherorganisms (prokaryotic or eukaryotic). These new DNA molecules can beisolated from such a library using the polynucleotide sequence disclosedherein with standard hybridization techniques or by the amplification ofsequences using polymerase chain reaction (PCR) amplification. Suitableprobes for use in identifying protein homolog sequences can be obtainedfrom gene-specific sequences. Alternatively, oligonucleotides containingspecific DNA sequences from coding region for these genes can be used toidentify related clones. One of ordinary skill in the art willappreciate that the regulatory regions of the genes and homologous genescan be obtained using similar methods.

Homologous polynucleotide molecules can be isolated using standardhybridization techniques with probes of at least about 7 nucleotides,more preferably 15 nucleotides, in length (but can be as much as thefull coding sequence). Homologous polynucleotide sequences can beidentified using degenerate oligonucleotides based on the sequencesdisclosed herein which are capable of hybridization at moderate orgreater stringency. The term, “capable of hybridization” as used hereinmeans that the subject nucleic acid molecules (whether DNA or RNA)anneal to an oligonucleotide of 15 or more contiguous nucleotides of oneof the polynucleotide sequences disclosed herein.

The choice of hybridization conditions will be evident to one ordinarilyskilled in the art and will generally be guided by the purpose of thehybridization, the type of hybridization (DNA-DNA or DNA-RNA), and thelevel of desired relatedness between the sequences. Methods forhybridization are well established in the literature. One of ordinaryskill in the art realizes that the stability of nucleic acid duplexesdecrease with an increased number and location of mismatched bases. As aresult, the stringency of hybridization can be used to maximize orminimize the stability of such duplexes. Hybridization stringency can bealtered, for example, by adjusting the temperature of hybridization,adjusting the percentage of helix-destabilizing agents (e.g., formamide)in the hybridization mix, and adjusting the temperature and saltconcentration of the wash solutions. In general, the stringency ofhybridization is adjusted during post-hybridization washes by varyingthe salt concentration and/or the temperature, which results inprogressively higher stringency conditions.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. Optimal conditions will vary depending on the particularhybridization reaction involved, and can be determined empirically.Conditions of high stringency are preferably used for the hybridizationof the probe of interest.

Alternatively, polynucleotides having substantially the same nucleotidesequence as or are substantially identical to one of the polynucleotidesequences of aap or the aat gene cluster represent aap-like or aat genecluster-like genes. Also polynucleotides which encode for proteins thatare functionally equivalent of, or functional fragments of Aap or theproteins of the aat gene cluster are included in this invention.“Substantially the same” or “substantially identical” is meant a nucleicacid, polynucleotide, or polypeptide exhibiting at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to areference nucleic acid, polynucleotide, or polypeptide. For nucleotidesequences, the length of comparison sequences will generally be at least10 to 500 nucleotides in length. Preferably, the length of comparisonwill be at least 50 nucleotides, more preferably at least 60nucleotides, even more preferably at least 75 nucleotides, and mostpreferably at least 110 nucleotides in length.

An “isolated” nucleic acid is a nucleic acid the structure of which isnot identical to that of any naturally occurring nucleic acid or to thatof any fragment of a naturally occurring genomic nucleic acid spanningmore than three separate genes. The term therefore covers, for example,(a) a DNA which has the sequence of part of a naturally occurringgenomic DNA molecule but is not flanked by both of the coding sequencesthat flank that part of the molecule in the genome of the organism inwhich it naturally occurs; (b) a nucleic acid incorporated into anexpression vector or into the genomic DNA of a prokaryote or eukaryotein a manner such that the resulting molecule is not identical tonaturally occurring expression vector or genomic DNA; (c) a separatemolecule such as cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; and (d) ahybrid gene, i.e., a gene encoding a fusion protein. Specificallyexcluded from this definition are nucleic acids present in mixtures of(i) DNA molecules, (ii) transfected cells, and (iii) cell clones, e.g.,as these occur in a DNA library such as a cDNA or genomic DNA library.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization or computer-basedtechniques that are well-known in the art. Such techniques include, butare not limited to, the hybridization of genomic or cDNA libraries withprobes to detect homologous nucleotide sequences; antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features; polymerase chain reaction (PCR) on genomic DNA orcDNA using primers capable of annealing to the DNA sequence of interest;computer searches of sequence databases for similar sequences; anddifferential screening of a subtracted DNA library.

Screening procedures that rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate oligonucleotide probe is available. Oligonucleotide probes,which correspond to a part of the sequences for aap or the aat genecluster which are provided herein, can be synthesized chemically.Synthesis of other oligonucleotide probes may require that short,oligo-peptide stretches of the amino acid sequence be known because theDNA sequence encoding a specific protein can be deduced using thegenetic code; however, the degeneracy of the code must be taken intoaccount. When the sequence is degenerate, it is possible to perform amixed addition reaction that includes a heterogeneous mixture ofdenatured double-stranded DNA. For such screening, hybridization ispreferably performed on either single-stranded DNA or denatureddouble-stranded DNA. Hybridization is particularly useful in thedetection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. By using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA or DNA clone by thehybridization of the target DNA to that single probe in the mixture thatis its complete complement. (Wallace et al., Nuc. Acid Res. 9:879(1981)). Alternatively, a subtractive library is useful for eliminationof non-specific cDNA clones.

Another standard procedure for isolating DNA sequences of interest isthe formation of plasmid- or phage-carrying genomic libraries whichinclude total DNA from the organism of interest. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target DNAcan be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the DNA that have been denatured into asingle-stranded form. (Jay et al., Nucl. Acid Res., 11:2325 (1983).

The nucleotide sequences of aap and the aat gene cluster have a myriadof applications. Representative uses of these nucleotide sequencesinclude the construction of DNA and oligonucleotide probes useful inNorthern, Southern, immuno-PCR, dot-blot, and other assays for detectingEAEC, quantifying the level of expression of Aap, AatP, AatA, AatB,AatC, or AatD in a cell, or generating the particular protein orpolypeptide which is encoded by the nucleotide sequence. aap and the aatgene cluster nucleotide sequences can be employed for the constructionof recombinant cell lines, recombinant organisms, expression vectors,and the like. Such recombinant constructs can be used to expressrecombinant Aap, AatP, AatA, AatB, AatC, and/or AatD, and can be used asvaccines (via a live, attenuated vector vaccine or other type ofinvasive vector vaccine) or to screen for candidate therapeutic agentscapable of altering the pathology of an organism expressing one or morethese proteins.

Considering the important role these proteins plays in EAEC aggregation,penetration of the mucosal mucous blanket, attachment, and colonization,the proteins or polypeptides of this invention are highly useful in thegeneration of immunogenic compositions that can be used to generate animmune response in a subject (e.g., via a subunit vaccine) and in kitsfor the detection of EAEC. For a subunit vaccine, one or more of theproteins of this invention can be expressed, purified, and used toprepare an immunogenic subunit composition. For a live, attenuatedvector vaccine, one or more of the proteins of this invention can alsobe expressed in an attenuated, invasive bacteria or virus, and the wholeorganism can be formulated into an immunogenic composition. In anothertype of vaccine, one or more of the genes of this invention can beplaced on a plasmid downstream of a signal sequence of an eukaryoticpromoter. That plasmid can contain one or more selectable markers and betransfected into a prokaryotic organism, such as Salmonella spp.,Shigella spp., or other suitable bacteria. The bacteria is thenadministered to the eukaryotic subject for which immune response to EAECis desired. See, for example, U.S. Pat. No. 5,887,159 to Hone, et al.Additionally, a polynucleotide encoding one of the proteins of thisinvention can be administered to the mucosal tissue of a subject togenerate an immunogenic response. See, for example, U.S. Pat. No.6,110,898 to Malone et al. Also, a naked polynucleotide encoding one ofthe proteins of this invention can be electroporated into a subject togenerate an immune response using the methods described within Drabick,J. J.; et al.; Cutaneous transfection and immune responses tointradermal nucleic acid vaccination are significantly enhanced by invivo electropermeabilization, Mol. Ther. Vol 3(2); pp. 249-55 (2001).

The genes of this invention can also be placed into expression vectorswhich are discussed more fully below.

Antisense

Antisense nucleotide sequences to the genes of this invention can beused to block expression of proteins of this invention. Suitableantisense oligonucleotides are at least 11 nucleotides in length and caninclude untranslated (upstream) and associated coding sequences. Asknown by those of skill in the art, the optimal length of an antisenseoligonucleotide depends on the strength of the interaction between theantisense oligonucleotide and the complementary mRNA, the temperatureand ionic environment in which translation takes place, the basesequence of the antisense oligonucleotide, and the presence of secondaryand tertiary structures in the mRNA and/or in the antisenseoligonucleotide. Suitable target sequences for antisenseoligonucleotides include promoter regions, ribosome binding sites, andsites that interfere with ribosome progression.

Antisense oligonucleotides can be prepared, for example, by inserting aDNA molecule containing the target DNA sequence into a suitableexpression vector such that the DNA molecule is inserted downstream of apromoter in a reverse orientation as compared to a particular gene ofthis invention. The expression vector can then be transduced,transformed, or transfected into a cell (prokaryotic and/or eukaryotic)suitable for expressing the antisense oligonucleotides. Alternatively,antisense oligonucleotides can be synthesized using standard manual orautomated synthesis techniques. These synthesized oligonucleotides areintroduced into suitable cells by a variety of means includingelectroporation, calcium phosphate precipitation, and microinjection.The selection of a suitable antisense oligonucleotide administrationmethod would be evident to one of ordinary skill in the art.

With respect to synthesized oligonucleotides, the stability of antisenseoligonucleotide-mRNA hybrids is advantageously increased by the additionof stabilizing agents to the oligonucleotide. One example of astabilizing agent includes intercalating agents that are covalentlyattached to either or both ends of the oligonucleotide. In preferredembodiments, the oligonucleotides are made resistant to nucleases bymodifications to the phosphodiester backbone by the introduction ofphosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates,phosphoramidates, phosphorodithioates, or morpholino rings.

Amino Acids

“Aap”, “AatP”, “AatA”, “AatB”, “AatC”, and “AatD”, as described herein,encompass the whole protein (Aap, AatP, AatA, AatB, AatC, and AatDrespectively), fragments of the protein (Aap, AatP, AatA, AatB, AatC,and AatD respectively) that are functionally active, and polypeptidesthat contain substitutions such as one basic amino acid for anotherbasic amino acid, or one acidic amino acid for another acid amino acid,or one neutral amino acid for another neutral amino acid. “Aap”, “AatP”,“AatA”, “AatB”, “AatC”, and “AatD” also encompass Aap, AatP, AatA, AatB,AatC, and AatD respectively purified from naturally occurring materialsand closely related, functionally similar proteins retrieved by antiseraspecific to the particular protein. Recombinantly expressed proteinsencoded by genetic materials (DNA, RNA, cDNA) retrieved on the basis oftheir similarity to regions in the particular gene sequence are alsoencompassed by the present description.

Polynucleotide molecules encoding Aap, AatP, AatA, AatB, AatC, and AatDinclude molecules that encode Aap, AatP, AatA, AatB, AatC, and AatD,respectively, or peptides that share identity with the sequence shown inSEQ ID NO: 2 (FIG. 2); SEQ ID NO: 4 (FIG. 10); SEQ ID NO: 5 (FIG. 11);SEQ ID NO: 6 (FIG. 12); SEQ ID NO: 7 (FIG. 13), and SEQ ID NO: 8 (FIG.14) respectively. These molecules preferably share greater than 30%identity at the amino acid level with the disclosed protein. Inpreferred embodiments, the polynucleotide molecules share greateridentity at the amino acid level across highly conserved regions.Variants of a particular gene encoded protein include those amino acidsequences resulting from minor genetic polymorphisms, differencesbetween strains, and those that contain amino acid substitutions,additions, and/or deletions, and conservative amino acid substitutions.A conservative amino acid substitution includes a replacement of oneamino acid residue with a different residue having similar biochemicalcharacteristics, such as size, charge, and polarity vs. nonpolarity.

Amino acid sequences substantially the same as the sequences set forthin SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7;and SEQ ID NO: 8 are encompassed by the present description. A preferredembodiment includes polypeptides having substantially the same sequenceof amino acids as the sequences described herein, functional fragmentsthereof, or amino acid sequences that are substantially identical to thesequences described herein. As described above, “substantially the same”or “substantially identical” is meant a polypeptide exhibiting at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, to 100%homology to a reference amino acid sequence. For polypeptides, thelength of comparison sequences will generally be at least 16 aminoacids, preferably at least 20 amino acids, more preferably at least 25amino acids, and most preferably at least 35 amino acids.

Homology of sequences is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705 or the NCBI BLAST program). Such softwarematches similar sequences by assigning degrees of homology to varioussubstitutions, deletions, substitutions, and other modifications.

The term “substantially identical” also means an amino acid sequencewhich differs only by conservative amino acid substitutions, forexample, substitution of one amino acid for another of the same class(e.g., valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative substitutions, deletions, or insertions located atpositions of the amino acid sequence that do not destroy the function ofthe protein assayed, (e.g., as described herein). Preferably, such asequence is at least 85%, and more preferably from 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, to 100% homologous at the amino acid levelto sequences described herein.

The term “functional fragments” include fragments of the proteinsdescribed herein that have a similar amino acid sequence and that retainthe function or activity of the particular protein. Where full-lengthprotein is described, one of skill in the art can screen for thefunctionality of a fragment by using the examples provided herein.

The term “substantially pure polypeptide” as used herein means anpolypeptide that has been separated from components that naturallyaccompany it. Typically, the polypeptide is substantially pure when itis at least 60%, by weight, free from the proteins and other naturallyoccurring molecules with which it is typically associated. Preferably,the preparation is at least 75%, 80%, 90%, 95%, and most preferably atleast 99%, by weight. A substantially pure protein can be obtained, forexample, by extraction from a natural source, by expression of arecombinant nucleic acid encoding a particular protein, or by chemicallysynthesizing the protein. Purity can be measured by any appropriatemethod, such as column chromatography, polyacrylamide gelelectrophoresis, and HPLC analysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants that accompany it in its naturalstate. Thus, a protein that is chemically synthesized or produced in acellular system different from the cell or host from which it naturallyoriginates is substantially free from its naturally associatedcomponents. Accordingly, substantially pure polypeptides include thosederived from prokaryotic organisms but synthesized in eukaryoticorganisms. A purified polypeptide is a polypeptide substantially free ofnaturally associated components when it is separated from thosecontaminants that accompany it in its natural state. As would be evidentto one ordinarily skilled in the art, the polynucleotide moleculesaccording to the present disclosure can be expressed in a variety ofprokaryotic and eucaryotic cells using regulatory sequences, expressionvectors, and methods well established in the literature to produce apolypeptide or protein which can be purified.

Purification of a protein of this invention can be performed using anumber of established methods such as affinity chromatography usingantibodies specific to a particular protein coupled to a solid support.Fusion proteins of an antigenic tag and the protein of interest can bepurified using antibodies to the tag. Optionally, additionalpurification is achieved using conventional purification means such asliquid chromatography, gradient centrifugation, or gel electrophoresis.Methods of protein purification are well known in the art and can beapplied to the purification of recombinant proteins described herein.The purification of the proteins of this invention is discussed in moredetail below.

Fusion proteins typically contain additions, substitutions, orreplacements of one or more contiguous amino acids of the native proteinwith amino acid(s) from a suitable fusion protein partner. Such fusionproteins are obtained using recombinant DNA techniques easily identifiedand well known by those of skill in the art. For example, DNA moleculesencoding the hybrid Aap fusion protein of interest are prepared usinggenerally available methods such as PCR mutagenesis, site-directedmutagenesis, and/or restriction digestion and ligation. The hybrid DNAis then inserted into expression vectors and introduced into suitablehost cells.

One embodiment of the present invention involves the isolation ofproteins that interact with the proteins described herein or arereceptors for the proteins described herein. Aap, AatP, AatA, AatB,AatC, and/or AatD can be used in immunoprecipitation to isolateinteracting factors or for the screening of interactors using differentmethods of two hybrid screening. Isolated interactors of these proteinscan be used to modify or block activity of the particular protein in ahost or between EAEC.

Synthetic peptides, recombinantly derived peptides, fusion proteins,chiral proteins (stereochemical isomers, racemates, enantiomers, andD-isomers), and the like are provided which include a portion of one ofthe proteins described herein or the entire protein. The subjectpeptides have an amino acid sequence encoded by a nucleic acid whichhybridizes under stringent conditions with an oligonucleotide of 15 ormore contiguous nucleotides of SEQ ID NO: 1; SEQ ID NO: 9; SEQ ID NO:10;SEQ ID NO: 11; SEQ ID NO: 12; or SEQ ID NO: 13. Representative aminoacid sequences of the subject peptides is disclosed in SEQ ID NO: 2; SEQID NO: 4, SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; or SEQ ID NO: 8. Thesubject peptides find a variety of uses, including preparation ofspecific antibodies and preparation of antagonists of activity theproteins described herein.

Antibodies that Bind To Aap, AatP, AatA, AatB, AatC, or AatD

The production of antisera or monoclonal antibodies (e.g., murine,lagomorph, porcine, equine, or human) is well known and can beaccomplished by methods easily identified by one of skill in the art,such as, for example, immunizing an animal with one of the proteinsdescribed herein or a peptide derived from one of the proteins. For theproduction of monoclonal antibodies, antibody producing cells areobtained from immunized animals, immortalized, and screened.Alternatively, antibody producing cells are first screened for theproduction of the antibody that binds to a particular protein or theparticular derived peptides and then immortalized. It can be desirableto transfer the antigen binding regions (e.g., F(ab′)2 or hypervariableregions) of non-human antibodies into the framework of a human antibodyby recombinant DNA techniques to produce a substantially human molecule.

Following synthesis or expression and isolation or purification of aparticular protein or a portion thereof, the isolated or purifiedprotein can be used to generate antibodies and tools for identifyingagents that interact with that particular protein and fragments ofinterest. Depending on the context, the term “antibodies” can encompasspolyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Antibodies thatrecognize Aap, AatP, AatA, AatB, AatC, and/or AatD and fragments thereofhave many uses including, but not limited to, biotechnologicalapplications, therapeutic/prophylactic applications, and diagnosticapplications.

For the production of antibodies, various hosts including, but notlimited to, goats, rabbits, rats, mice, humans, etc., can be immunizedby injection with a particular protein or any portion, fragment, oroligopeptide that retains immunogenic properties. Depending on the hostspecies, various adjuvants can be used to increase the immunologicalresponse. Such adjuvants include, but are not limited to, detoxifiedheat labile toxin from E. coli, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacteriumparvum are also potentially useful adjuvants.

Peptides used to induce specific antibodies have an amino acid sequenceof at least three amino acids, and preferably an amino acid sequence ofat least 10 to 15 amino acids. Preferably, short stretches of aminoacids encoding fragments of a particular protein are fused with those ofanother protein such as keyhole limpet hemocyanin such that an antibodyis produced against the chimeric molecule. Although antibodies capableof specifically recognizing a particular protein of this invention canbe generated by injecting synthetic 3-mer, 10-mer, and 15-mer peptidesthat correspond to an amino acid sequence of that particular proteininto a host (e.g., mice), a more diverse set of antibodies can begenerated by using a particular recombinant protein, a particularpurified protein, or fragments of a particular protein.

To generate antibodies to Aap, AatP, AatA, AatB, AatC, AatD, and/orfragments thereof, a substantially pure Aap, AatP, AatA, AatB, AatC,AatD, or a fragment thereof is isolated from a transfected ortransformed cell or the wildtype EAEC. The concentration of thepolypeptide in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms/ml.

Monoclonal antibodies to Aap, AatP, AatA, AatB, AatC, AatD, or afragment thereof can be prepared using any technique that provides forthe production of antibody molecules by continuous cell lines inculture. Such techniques include, but are not limited to, the hybridomatechnique originally described by Koehler and Milstein (Nature256:495-497 (1975)), the human B-cell hybridoma technique (Kosbor etal., Immunol Today 4:72 (1983); Cote et al., Proc Natl. Acad. Sci80:2026-2030 (1983)), and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New YorkN.Y., pp 77-96 (1985)). Techniques developed for the production of“chimeric antibodies”, i.e., the splicing of mouse antibody genes tohuman antibody genes to obtain a molecule with appropriate antigenspecificity and biological activity, can also be used. (Morrison et al.,Proc Natl. Acad. Sci 81:6851-6855 (1984); Neuberger et al., Nature312:604-608(1984); Takeda et al., Nature 314:452-454(1985)).Alternatively, techniques described for the production of single chainantibodies, such as disclosed in U.S. Pat. No. 4,946,778, incorporatedherein by reference in its entirety, can be adapted to produceAap-specific single chain antibodies. Additionally, antibodies can beproduced by inducing in vivo production in the lymphocyte population orby screening recombinant immunoglobulin libraries or panels of highlyspecific binding reagents as disclosed in Orlandi et al., Proc Natl.Acad. Sci 86: 3833-3837 (1989); and Winter G. and Milstein C., Nature349:293-299 (1991).

Antibody fragments that contain specific binding sites for a particularprotein of this invention can also be generated. For example, suchfragments include, but are not limited to, F(ab′)₂ fragments produced bypepsin digestion of the antibody molecule and Fab fragments generated byreducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively,Fab expression libraries can be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.(Huse W. D. et al., Science 256:1275-1281 (1989)).

One method of making monoclonal antibodies to Aap, AatP, AatA, AatB,AatC, AatD, or fragments thereof includes repetitively inoculating amouse with a few micrograms of the selected protein or peptides over aperiod of a few weeks. The mouse is then sacrificed and the antibodyproducing cells of the spleen are isolated. These spleen cells are fusedin the presence of polyethylene glycol and mouse myeloma cells. Theexcess unfused cells are destroyed by growth of the system on selectivemedia comprising aminopterin (HAT media). The successfully fused cellsare then diluted and aliquots of the dilution are placed in wells of amicrotiter plate where growth of the culture is continued.Antibody-producing clones are identified by the detection of antibody inthe supernatant fluid of the wells by immunoassay procedures such asELISA, as originally described by Engvall, E., Meth. Enzymol. 70:419(1980), and derivative methods thereof. Selected positive clones can beexpanded and their monoclonal antibody product harvested for use. Anexample of a detailed procedure for monoclonal antibody production canbe found in Davis, L. et al., Basic Methods in Molecular BiologyElsevier, New York. Section 21-2.

Polyclonal antiserum containing antibodies to heterogeneous epitopes ofa single protein can be prepared by immunizing suitable animals with theexpressed protein or peptides derived therefrom, which can be unmodifiedor modified to enhance immunogenicity. Effective polyclonal antibodyproduction is affected by many factors related both to the antigen andto the host species. For example, small molecules tend to be lessimmunogenic than larger molecules and can require the use of carriersand adjuvants. Also, host animals vary in response to the site(s) ofinoculations and dose, with both inadequate or excessive doses ofantigen resulting in low titer antisera. Small doses (e.g., ng level) ofantigen administered at multiple intradermal sites appears to be mostreliable. An example of an effective immunization protocol for rabbitscan be found in Vaitukaitis, J. et al., J. Clin. Endocrinol. Metab.33:988-991 (1971).

Booster injections can be given at regular intervals, and antiserumharvested when the antibody titer thereof begins to fall. This fall inantibody titer can be determined semi-quantitatively, for example, bydouble immunodiffusion in agar against known concentrations of theantigen. (See, for example, Ouchterlony, O. et al., Chap. 19 in:Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973)).Plateau concentration of antibody is usually in the range of 0.1 to 0.2mg/ml of serum (about 12 μM). Affinity of the antisera for the antigenis determined by preparing competitive binding curves, as described, forexample, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2dEd. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington,D.C. (1980). Antibody preparations prepared according to either protocolare useful in quantitative immunoassays that determine concentrations ofantigen-bearing substances in biological samples. These antibodypreparations are also used semi-quantitatively or qualitatively (e.g.,in diagnostic embodiments that identify the presence of Aap inbiological samples). It is also contemplated that various methods ofmolecular modeling and rational drug design can be applied to identifycompounds that resemble Aap, AatP, AatA, AatB, AatC, AatD, fragments, orderivatives thereof, and molecules that interact with Aap, AatP, AatA,AatB, AatC, and/or AatD, and, thereby modulate the particular protein'sfunction.

Non-therapeutic uses of antibodies to Aap or Aat proteins would includedetection of the bacteria. Monoclonal or polyclonal antibodies could beused in slide agglutination assays to rapidly detect the bacteria. E.coli from a stool would be passaged onto nutrient agar and cultivatedovernight at 37° C. Colonies would then be picked with a toothpick andresuspended in one drop of phosphate buffered saline on a glassmicroscope slide. One drop of antibody suspension would then be added tothe bacterial suspension, mixed and rocked for one minute. At this time,the suspension would be examined for agglutination of the bacteria.Agglutination would be indicative of a positive interaction ofantibodies and bacteria and would indicate that the bacteria expressedAap or Aat proteins. As a modification of this technique, the antibodiescould be affixed to latex beads to improve visualization.

Expression Vectors

Recombinant gene expression vectors containing any of the genes of thisinvention, or portions thereof, can be constructed in a variety of formswell-known in the art. Preferred expression vectors include plasmids andcosmids. Expression vectors include one or more fragments of aparticular gene and preferably comprise the full length gene. Anexpression vector containing one or more of the genes of this inventioncan be used to transfect or transform a suitable host cell (prokaryoticor eukaryotic) to produce the protein or to produce an immune response,or for some other purpose.

As used herein, the phrase “operatively encode” refers to one or moreprotein coding regions associated with those regulatory sequencesrequired for expression of the polypeptide encoded by the coding region.Examples of such regulatory regions include promoter binding sites,enhancer elements, ribosome binding sites, and the like. Those ofordinary skill in the art will be able to select regulatory sequencesand incorporate them into the recombinant expression vectors describedherein without undue experimentation. For example, suitable regulatorysequences for use in various eukaryotic and prokaryotic systems aredescribed in Ausubel, et al., Short Protocols in Molecular Biology,3^(rd) ed., John Wiley & Sons, Inc, New York, 1997, which is herebyincorporated by reference in its entirety.

Expression vectors for use with the aap gene or the aat gene clustertypically contain regulatory sequences derived from a compatible speciesfor expression in the desired host cell. For example, when E. coli isthe host cell, the host cell population is typically transformed usingpBR322, a plasmid derived from an E. coli species that contains genesfor ampicillin (AMPR) and tetracycline resistance. (Bolivar, et al.,Gene 2:95 (1977)). Thus, pBR322 provides an easy means for identifyingtransformed cells. The plasmids described in Galen, WO 00/32047 are alsouseful for expression vectors in some bacteria.

Promoters suitable for use with prokaryotic hosts illustratively includethe beta-lactamase and lactose promoter systems (Chang, et al., Nature275:617 (1978); Goeddel, et al., Nature 281:544 (1979)), alkalinephosphatase, the tryptophan (trp) promoter system (Goeddel, NucleicAcids Res. 8:4057 (1980)), ompC, nirB, and hybrid promoters such as thetaq promoter (de Boer, et al., Proc. Natl. Acad. Sci. USA 80:21-25(1983)). Other functional bacterial promoters are also suitable, andwould be easily identifiable by those of skill in the art. Thenucleotide sequences of these functional bacterial promoters are alsogenerally known in the art, thereby enabling a skilled worker to ligatethem to a polynucleotide encoding the peptide of interest (Siebenlist,et al., Cell 20:269 (1980)) using linkers or adapters to supply anyrequired restriction sites.

Eukaryotic microbes such as yeast cultures can also be used as sourcesfor the regulatory sequences. For example, Saccharomyces cerevisiae is acommonly used eukaryotic host microorganism. Suitable promotingsequences for use with yeast hosts include the promoters for3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem. 255:12073(1980)) or other glycolytic enzymes such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase (Hess, et al. J. Adv. EnzymeReg. 7:149 (1968); Holland, Biochemistry 17:4900 (1978)).

Other yeast promoters, which are inducible promoters having theadvantage of transcription controlled by growth conditions, include thepromoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degraded enzymes associated with nitrogen metabolism,metallothionine, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Yeast enhancers areadvantageously used with yeast promoters.

A recombinant virus can also be used as the expression vector. Exemplaryviruses include the adenoviruses, adeno-associated viruses, herpesviruses, vaccinia, CMV, BLUESCRIPT (Stratagene, San Diego, Calif.),baculovirus, or an RNA virus such as a retrovirus or an alphavirus.Preferably, the retroviral vector is a derivative of a murine or avianretrovirus. The alphavirus vector is preferably derived from Sindbis orSemliki Forest Virus. All of these expression vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated.

The viral vector can be made target specific by inserting one or moresequences of interest into the viral vector, along with another genewhich encodes the ligand for a receptor on a specific target cell. Forexample, retroviral vectors can be made target specific by inserting apolynucleotide encoding a sugar, a glycolipid, or a protein. Preferredtargeting is accomplished by using an antibody to target the retroviralvector, such as to the vicinity of a mucosal inductor site, using aMALT-specific antibody. Those of skill in the art will know of, or canreadily ascertain without undue experimentation, specific polynucleotidesequences which can be inserted into the retroviral genome to allowtarget specific delivery of the retroviral vector containing thepolynucleotides of interest.

In some instances, it can be preferable to use a selectable marker toidentify cells or organisms that contain the expression vector and theDNA of interest. Selectable markers are generally introduced into thecells or organisms along with the cloned DNA molecules and include genesthat confer resistance to drugs such as ampicillin, neomycin,hygromycin, and methotrexate. Selectable markers can also complementauxotrophies in the host cell, or can provide for detectable signals,such as beta-galactosidase, green fluorescent protein, or yellowfluorescent protein, to identify cells or organisms containing thecloned DNA molecules.

It will be appreciated that the same techniques that are utilized toincorporate the nucleotide sequences of aap and/or the aat gene cluster,and optionally other immunostimulatory polynucleotides, into viral geneexpression vectors can be used to incorporate the sequences into liveand attenuated live viruses for use as immunogenic compositions.

Targeting of mucosal tissues can be performed by exploiting inherentbiological properties of the lymphoid bed which is to be targeted. Theseproperties include the crypt architecture of the tonsillar pillars whichcan be used to entrap particles and also include the M cells of Peyer'spatches in the gut which specifically endocytose a wide variety ofparticles including lipid particles and other small particulates.Therefore, those skilled in the art can prepare a wide variety ofmolecular particulate preparations which, if provided to the intestine,will lodge within the crypt portions of intestinal Peyer's patches andbe endocytosed by M cells. If such particles provide for delivery of abiologically active polynucleotide to M cells, the particles will enablethe stimulation or modulation of a mucosal immune response induction bythe Peyer's patch lymphoid tissue to which the M cell traffics.

Construction of suitable expression vectors containing desired coding,non-coding, and control sequences employ standard ligation techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to construct the required plasmids. To confirmcorrect sequences in the plasmids constructed, the ligation mixtures canbe used, for example, to transform a host cell and successfultransformants selected by antibiotic resistance where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionand/or sequenced by, for example, by the method disclosed in Messing, etal. (Nucleic Acids Res., 9:309 (1981)), Maxam, et al. (Methods inEnzymology 65:499 (1980)), or other suitable methods which will be knownto those skilled in the art. Size separation of cleaved fragments can beperformed using conventional gel electrophoresis as described, forexample, by Maniatis, et al. (Molecular Cloning, pp. 133-134 (1982)).

Host cells can be transformed with the expression vectors describedherein and cultured in conventional nutrient media modified as isappropriate for inducing promoters, selecting transformants, oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

Purification

Steps involved in the purification of one or more of the proteins ofthis invention include (1) solubilization of the desired protein, (2)the development of one or more isolation and concentration procedures,(3) stabilization of the protein following purification, and (4)development of a suitable assay to determine the presence of the desiredprotein. Various aspects of protein isolation and purification arediscussed in detail in Cooper, T. G., “The Tools of Biochemistry,” JohnWiley & Sons, New York, 1977, which is hereby incorporated by referencein its entirety. As the techniques of protein isolation and purificationare notoriously well known in the art, this disclosure will refrain fromdiscussing them in detail. Nevertheless, elements of the cited referenceare summarized and discussed below.

Solubilization is required of most proteins that are to be purified, asmost isolation procedures commonly used operate in aqueous solutions. Insome cases, solubilization can be achieved by merely lysing a host cellwithin which a desired protein has been expressed. In other situations,additional steps, such as extracting the desired protein from asubcellular organelle, may be required. Osmotic lysis, grinding, the useof blenders, ultrasonic waves, presses, and other well known techniquesof protein solubilization can be used with the methods disclosed herein.

There are a variety of techniques available that are well known in theart for the isolation and concentration of the proteins of thisinvention. These techniques include, but are not limited to, (1)differential solubility, (2) ion exchange chromatography, (3) absorptionchromatography, (4) molecular sieve techniques, (5) affinitychromatography, (6) electrophoresis, and (7) electrofocusing. Each ofthese techniques can also be useful in the purification of a protein ofthis invention. An example of the purification of Aap can be foundbelow.

Stabilizing and maintaining a purified protein product in a functionalstate warrants attention to a number of different conditions such as (1)pH, (2) degree of oxidation, (3) heavy metal concentration, (4) mediumpolarity, (5) protease concentration, and (6) temperature. One ofordinary skill in the art would readily know which of the availabletechniques to use to maintain purified protein in an active form withoutundue experimentation.

Compositions

The proteins encoded by aap and the aat gene cluster can be used toformulate immunogenic compositions that facilitate an immune response.Examples of a typical immune response to EAEC infections include amucosal immune response and a systemic immune response.

In accordance with one aspect of the present invention, smallerfragments of the proteins of this invention are used to provide animmunogenic composition. Specifically, these fragments comprise animmunogenic region of expression products, typically from about 5, 6, 8,10 or 12 amino acids to about 20, 22, 24, 30, or more amino acids.Suitable fragments or immunogenic regions can be readily ascertainedusing the techniques set forth below as screening procedures. In oneexample of a suitable screening procedure, a large number of candidatefragments are more or less randomly produced and used to immunize guineapigs or other suitable models. Alternatively, full-length polypeptidesshown to be active in the present invention can be truncated andscreened in an iterative process to isolate the immunogenic andprotective activity to a minimal fragment. Such screening can be readilycarried out without undue experimentation and the active fragments arewithin the contemplation of the present invention. A third example wouldbe to predict surface-exposed and immunogenic epitopes usinghydrophobicity and/or other amino acid properties. Predictedsurface-exposed residues would be synthesized as oligopeptides andantibodies would be raised against these peptides, e.g., in a rabbit ormouse. The antibodies would then be tested for reaction against theintact protein. Binding to the protein would suggest that the predictedepitopes are indeed surface-exposed and could therefore representpotentially protective epitopes for an EAEC vaccine.

Recombinant Organisms

In one embodiment of the present invention, a nucleotide sequencecomprising the aap gene or a functional fragment thereof or one or moreof the genes or fragments thereof of the aat gene cluster is introducedinto an exogenous organism using standard molecular biology techniqueswell known to those of ordinary skill in the art. Exemplary molecularbiology techniques are discussed in Ausubel, et al., “Short Protocols inMolecular Biology.” The resulting recombinant organism can then be usedas an immunogen against which an immune response may be engendered. In apreferred embodiment, an attenuated pathogenic organism serves as theexogenous organism. In addition, it is contemplated that an entirerecombinant organism or a functional fragment thereof, such as anisolated membrane fraction, liposome, or the like, can be used togenerate an immunogenic composition. One this contemplated embodiment,the aap gene would be co-expressed with the usher and chaperone from theparent organism, EAEC 042, to ensure secretion of the Aap proteinthrough the bacterial outer membrane. The usher (aafC) and chaperonegenes (aaJD) are available as chimeric clones. In another embodiment,the entire aat gene cluster would be expressed within an organism.

Subunit Immunogenic Compositions

Another embodiment of the present invention relates to the generation ofimmunogenic compositions comprising distinct immunogenic proteins (orfragments thereof) or functional fragments of the organisms of interest.Such immunogenic compositions are referred to herein as subunitimmunogenic compositions because at least one of the components of thecomposition is a subunit of an organism, rather than an entire organism.Typically, such a subunit immunogenic composition comprises one or moreimmunogenic components.

In a preferred embodiment, the subunit immunogenic composition includesa carrier component and an immunogenic component. Typically, the carriercomponent functions as a binding moiety with which the originatingorganism uses to bind to and gain entrance into the host organism. Inanother preferred embodiment, Aap, AatP, AatA, AatB, AatC, and/or AatDfrom EAEC functions as the carrier component. Any protein, peptide, oramino acid sequence that elicits an immune response can be used as theimmunogenic component in a subunit immunogenic composition.

The carrier component of the subunit immunogenic composition can possessimmunogenic characteristics themselves. Typically, adjuvants are used inimmunogenic compositions to enhance the immune response directed againstthe immunogenic component of the immunogenic compositions. Carriercomponents that possess both mucosa binding characteristics andimmunogenic characteristics can be used. For example, Aap, AatP, AatA,AatB, AatC, and/or AatD can function both as the carrier component andthe immunogenic component.

For the carrier components described above, the entire molecule can beused as the carrier component, or a functionally active fragment of themolecule can be used. Mutagenized forms of these molecules can also beused as carrier components.

Although not wishing to be bound by theory, it is hypothesized that thesubunit immunogenic composition functions by exposing the immunogeniccomponent of the subunit immunogenic composition to the mucosa and thevarious immune system components present there. According to one theory,the generation of a desired immune response by the subunit immunogeniccomposition occurs by increasing the exposure of the immunogeniccomposition to the target tissue. The presence of both a carriercomponent and an immunogenic component are theorized to achieve thisgoal.

In one embodiment of the instant invention, Aap is isolated, purified,and mixed or coupled with one or more immunogenic or adjuvant compounds.For example, Aap can be expressed, purified, and cross-linked to atoxin, toxoid (an attenuated toxin), or some other immunogenic compoundfor use in a subunit immunogenic composition. In another embodiment,AatA can be expressed, purified, and cross-linked to a toxin, toxoid (anattenuated toxin), or some other immunogenic compound for use in asubunit immunogenic composition.

In another embodiment, expressed Aap or fragments thereof or AatA orfragments thereof can be cross-linked to an immunogenic component, whichcan be an isolated protein, a functional fragment thereof, a wholeorganism (such as a bacterium or a virus) or functional fragment thereofthat is isolated either in part or is used as a whole pathogenicorganism. Aap and AatA can be isolated from the bacterium itself or itcan be produced using recombinant DNA techniques well known in the art.It is contemplated, and within the purview of the present invention,that an entire recombinant organism or a functional fragment thereof,such as an isolated membrane fraction, liposome, or the like, can beused to generate an immunogenic composition.

The proteins of this invention which are used to form the subunitimmunogenic compositions can be the whole protein, an immunogenicfragment thereof, a mutagenized form of the protein, or a fusion proteincomprising the particular protein or a fragment thereof and a suitablefusion partner (e.g., any protein, peptide or amino acid sequence thatfacilitates the expression and/or purification of the protein and fusionpartner using recombinant DNA techniques known in the art).Alternatively, one or more additional immunogens can serve as the fusionpartner in a fusion protein.

Nucleotide Immunogenic Compositions

An immune response can also be elicited using nucleotide-containingcompositions. For example, in one embodiment encompassed by the presentinvention, a mucosal or systemic immune response is elicited in a hostby administering an antigen-encoding polynucleotide preparationincluding DNA or RNA that encodes an antigenic epitope to the host.Preferably, the nucleotide-containing composition is administered to amucosal inductor site in the mucosal tissue of the host. Naked DNA maybe administered directly to the mucosa (e.g., in saline drops) or in arecombinant gene expression vector. Preferably, the recombinant geneexpression vector is not capable of replication or dissemination.

Nucleotide-containing immunogenic compositions also include live viralimmunogenic compositions. The viruses for use in the viral immunogeniccompositions include immunostimulatory polynucleotides. Preferably, atarget protein antigen is administered through its expression by arecombinant gene expression vector.

U.S. Pat. No. 6,110,898, to Malone, et al., entitled, “DNA vaccines foreliciting a mucosal immune response,” which is hereby incorporated byreference in its entirety, provides detailed teaching for the generationof such immunogenic compositions. In particular, Malone teachesobtaining a recombinant alphavirus vector system as described in Malone,J. G., et al., “Mucosal immune responses associated with polynucleotidevaccination”, Behring Inst Mitt 98:63-72 (1997 February). DNA encodingAap (for example) is substituted for the lacZ gene in the vector. Thereplication defective alphavirus particles are activated by combiningone volume of virus stock to 1/20 volume of Chymotrypsin 10 mg/ml (inPBS with Ca²⁺/Mg²⁺) and 1/50 volume of CaCl₂ (50 mM). This mixture isallowed to incubate at room temperature for thirty minutes and then puton ice. Next, a ½ volume of Aprotinin 2 mg/ml is added. The solution iskept on ice for up to one hour and discarded if not used.

Balb-C mice (SPF female, 6 week, Charles River) are inoculated with 106virion particles (10⁷/ml) via either intratracheal, intranasal, orintravenous routes. Animals are anesthetized with a cocktail of ketamine(22 mg/kg), xylazine (2.5 mg/kg) and acepromazine (0.75 mg/kg) prior toinoculation. Intratracheal inoculation is performed by making a smallmedial cut through the skin at the ventral site of the neck. Salivaryglands are teased apart using blunt dissection to expose the trachea.With the trachea visualized, a 30.5 gauge needle with a 1 cc tuberculinsyringe attached is placed through the rings of the trachea toward thebronchi. 100 μl of the above virion particles are injected into thelung. Intranasal installation consists of placing 50 μl of the virionparticles into one of the nares. Once this is taken into the nasalpassages by inhalation, the other side is inoculated in the same manner.Intravenous inoculation is performed using a 30.5 gauge needle with 100μl of the above virion particles inserted into the tail vein.

Blood is collected from the retro-orbital venous plexus at day 0, 14,and 28, using a microcapillary tube. The blood is allowed to sit at roomtemperature for 2-4 hours to clot, then it is spun in a IEC Centra MP4Rcentrifuge at 6,000 RPM for six minutes. The supernatant (serum) isremoved and stored at −20° C. until ELISA assays are performed.

Lung lavages at week 4 are performed by the following method. Mice arekilled by carbon dioxide asphyxiation. The ventrum is skinned and themesentery removed which exposes the liver. The liver is moved aside tovisualize the diaphragm. The diaphragm is opened and the rib cage is cutbi-laterally up through the sternum. This section is lifted up over themouse's head, leaving the trachea exposed. A transverse cut of thetrachea is made approximately 2 cm above the bronchus. A blunt 24 gaugeneedle is inserted 0.5 cm into the trachea and tied in place withsurgical thread. One ml of BBS (89 mM boric acid, 90 mM NaCl, pH 8.3[NaOH]) is slowly introduced into the lungs using a 1 ml tuberculinsyringe attached to the blunt needle and then the volume is slowlywithdrawn. The solution is centrifuged to produce a cellular(particulate) and a supernatant component. Recovered volumes arenormally in the range of 0.85 to 0.95 ml. This cellular and supernatantcomponents are stored at −20° C. until used.

Animals for which histological sections are to be taken are killed bycarbon dioxide on day two. Their lungs are fixed in paraformaldehyde forthirty minutes, the tissue is then incubated overnight at 4° C. in a mixof PBS+2 mM MgCl₂+30% sucrose. Lung tissue are cryosectioned and placedon gelatinized slides. The slides are then fixed and stained for Aapusing an biotinylated labeled anti-Aap antibody.

ELISA assays are performed on samples using 96 well microtiter platesusing the same basic protocol as described in by Engvall, E., Meth.Enzymol. 70:419 (1980), and derivative methods thereof. In brief, Aap issuspended to a concentration of 1 mg/ml with PBS supplemented with 5mg/ml bovine serum albumin. The wells of the microtiter plate are coatedwith 5 mg of Aap (in that solution) per ml BBS (89 mM boric acid, 90 mMNaCl, pH 8.3 [NaOH]). The plates are incubated overnight at 4° C. Theplates are dried by pounding on a stack of paper towels and blocked with150 μl BB (BBS as above with 1% bovine serum albumin added). After theplates sit at room temperature for 2 hours, eight two-fold dilutions ofsample sera in BB are pipetted into the microtiter plates (50 μl perwell). Dilutions are performed as follows: serum IgG and IgA—1:10 to1:5120, lavage IgG and IgA—1:1 to 1:128. The plates are incubatedovernight at 4° C. Plates are washed 5-6 time with BBS plus 0.05% Tweenand dried. Either alkaline phosphatase conjugated goat anti-mouse IgG at1:2000 dilution or goat anti-mouse IgA at 1:1000 dilution in BB areadded (50 μl per well). Plates are incubated for two hours at roomtemperature. They are washed and dried as described above and thesubstrate buffer (1 mg/ml p-nitrophenol phosphate, 50 mM Na-bicarbonatebuffer, pH 9.8, 1 mM MgCl₂) is added. Plates are incubated again at roomtemperature for one hour. A Dynatech MR5000 ELISA plate reader with a405 nm wavelength (Dynatech Laboratories, Chantilly, Va.) hooked up to aiMac (having the appropriate software) reads the plates. Backgroundsignal is defined using control serum, with positive titer identifiedat >2.5× background. For lavage samples, OD₄₀₅ is reported using the 1:2dilution, with positive signal defined as 2.5× background.

The results of the lung lavage studies demonstrate that intranasalinoculation results in high levels of both IgG and IgA (indicative ofmucosal immunity). Intratracheal and intravascular inoculations producea systemic immune response.

Alternatively, one or more of the genes of the aat cluster can beintroduced to an attenuated EAEC, Salmonella spp., Shigella spp.,Lactobacillus spp., or other attenuated bacteria which is invasive formucosal tissue, which then expresses the particular Aat protein encodedby the gene. The bacteria is administered to an animal to generate animmune response to the particular Aat protein encoded.

Formulations and Administration

The immunogenic compositions described herein can be formulated in avariety of useful formats for administration by a variety of routes.Concentrations of the immunogenic components in the formulationsdescribed will be such that an effective dose of the immunogeniccomponents is included in the formulation. Determination of such aconcentration would be readily apparent to those of ordinary skill inthe art.

Administration of the immunogenic compositions can be by nasalapplication, by inhalation, ophthalmically, orally, rectally, vaginally,or by any other mode that results in the immunogenic compositioncontacting mucosal tissues.

Solid formulations of the compositions for oral administration maycontain suitable carriers or excipients, such as corn starch, gelatin,lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol,dicalcium phosphate, calcium carbonate, sodium chloride, or alginicacid. Disintegrators that can be used include, without limitation,micro-crystalline cellulose, cornstarch, sodium starch glycolate, andalginic acid. Tablet binders that may be used include acacia,methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone,hydroxypropyl methylcellulose, sucrose, starch, and ethylcellulose.Lubricants that may be used include magnesium stearates, stearic acid,ailicone fluid, talc, waxes, oil, and colloidal silica.

In one embodiment of the present invention, the immunogenic compositionexists as an atomized dispersion for delivery by inhalation. Theatomized dispersion of the immunogenic components typically containscarriers common for atomized or aerosolized dispersions, such asbuffered saline and/or other compounds well known to those of skill inthe art. The delivery of the immunogenic compositions via inhalation hasthe effect of rapidly dispersing the immunogenic components to a largearea of mucosal tissues as well as quick absorption by the blood forcirculation of the immunogenic components. One example of a method ofpreparing an atomized dispersion is described in U.S. Pat. No.6,187,344, entitled, “Powdered Pharmaceutical Formulations HavingImproved Dispersibility,” which is hereby incorporated by reference inits entirety.

The immunogenic compositions described herein can also be formulated inthe form of a rectal or vaginal suppository. Typical carriers used inthe formulation of the inactive portion of the suppository includepolyethylene glycol, glycerine, cocoa butter, and/or other compoundswell known to those of skill in the art. Although not wishing to bebound by theory, delivery of immunogenic compositions via a suppositoryis hypothesized to have the effect of contacting a mucosal surface withthe immunogenic compositions for release to proximal mucosal tissues.Distal mucosal tissues also receive the immunogenic composition bydiffusion. Other suppository formulations suitable for delivery of theimmunogenic compositions encompassed by the present invention are alsocontemplated.

Additionally, immunogenic compositions also exist in a liquid form. Theliquid can be for oral dosage, for ophthalmic or nasal dosage as drops,or for use as an enema or douche. When the immunogenic composition isformulated as a liquid, the liquid can be either a solution or asuspension of the immunogenic composition. There are a variety ofsuitable formulations for the solution or suspension of the immunogeniccomposition that are well know to those of skill in the art, dependingon the intended use thereof. Liquid formulations for oral administrationprepared in water or other aqueous vehicles may contain varioussuspending agents such as methylcellulose, alginates, tragacanth,pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinylalcohol. The liquid formulations may also include solutions, emulsions,syrups and elixirs containing, together with the active compound(s),wetting agents, sweeteners, and coloring and flavoring agents. Variousliquid and powder formulations can be prepared by conventional methodsfor inhalation into the lungs of the mammal to be treated.

Delivery of the described immunogenic compositions in liquid form viaoral dosage exposes the mucosa of the gastrointestinal and urogenitaltracts to the immunogenic compositions. A suitable dose, stabilized toresist the pH extremes of the stomach, delivers the immunogeniccompositions to all parts of the gastrointestinal tract, especially theupper portions thereof. Any method of stabilizing the immunogeniccompositions in a liquid oral dosage such that the effective delivery ofthe composition is distributed along the gastrointestinal tract arecontemplated for use with the immunogenic compositions described herein.

Delivery of the described immunogenic compositions in liquid form viaophthalmic drops exposes the mucosa of the eyes and associated tissuesto the immunogenic compositions. A typical liquid carrier for eye dropsis buffered and contains other compounds well known and easilyidentifiable to those of skill in the art.

Delivery of the described immunogenic compositions in liquid form vianasal drops exposes the mucosa of the nose and sinuses and associatedtissues to the immunogenic compositions. Liquid carriers for nasal dropsare typically various forms of buffered saline.

A colloidal dispersion system may be used for targeted delivery ofnucleic acid-containing immunogenic compositions. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. A preferred colloidal system isa lipid preparation including unilamaller and multilamellar liposomes.

Injectable formulations of the compositions may contain various carrierssuch as vegetable oils, dimethylacetamide, dimethylformaamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, and liquid polyethylene glycol) and thelike. For intravenous injections, water soluble versions of thecompounds may be administered by the drip method, whereby apharmaceutical formulation containing an antifungal agent and aphysiologically acceptable excipient is infused. Physiologicallyacceptable excipients may include, for example, 5% dextrose, 0.9%saline, Ringer's solution or other suitable excipients. Intramuscularpreparations (e.g., a sterile formulation of a suitable soluble saltform of the composition) can be dissolved and administered in apharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5%glucose solution. A suitable insoluble form of the composition may beprepared and administered as a suspension in an aqueous base or apharmaceutically acceptable oil base, such as an ester of a long chainfatty acid (e.g., ethyl oleate).

Liposomes are artificial membrane vesicles that are useful as deliveryvehicles in vitro and in vivo. It has been shown that large unilamellarvesicles (LUV), which range in size from 0.2-4.0 μm, can encapsulate asubstantial percentage of an aqueous buffer containing largemacromolecules. RNA, DNA, and intact virions can be encapsulated withinthe aqueous interior and can be delivered to cells in a biologicallyactive form (Fraley, et al., Trends Biochem. Sci. 6:77 (1981)). Inaddition to mammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast, and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genesencoding the polynucleotides at high efficiency while not compromisingtheir biological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation. (Mannino, et al., Biotechniques 6:682 (1988)). In additionto such LUV structures, multilamellar and small unilamellar lipidpreparations that incorporate various cationic lipid amphiphiles canalso be mixed with anionic polynucleotides to form nucleolipidicparticles which are often also referred to as liposomes. (Felgner, etal., Proc Natl. Acad. Sci. U.S.A. 84 (21): 7413 (1987)). Thesenucleophilic particles can be used to deliver the nucleic acids intocells.

The composition of the liposome is usually a combination ofphospholipids, preferably high-phase-transition-temperaturephospholipids, usually in combination with steroids, preferablycholesterol. However, other phospholipids or other lipids may also beused. The physical characteristics of the liposomes depend on pH, ionicstrength, and the presence of divalent cations. The appropriatecomposition and preparation of cationic lipid amphiphile:polynucleotideformulations are known to those skilled in the art, and a number ofreferences which provide this information are available (e.g., Bennett,et al, J. Liposome Res. 6(3):545).

Examples of lipids useful in liposome production include, but are notlimited to phosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, preferably from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.Examples of cationic amphiphilic lipids useful in formulation ofnucleolipid particles for polynucleotide delivery include the monovalentlipids DOTAP, DOTMA, and DC-Chol, the polyvalent lipids LipofectAMINE,DOGS, Transfectam, and other amphiphilic polyamines. These agents may beprepared with helper lipids (such as Dioleoyl Phosphatidyl Ethanolamine)or with various carrier compositions, including various adjuvants suchas cholera-derived molecules including cholera toxin.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs that contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used to join the lipidchains to the targeting ligand.

Administration of the compounds discussed above can be practiced invitro or in vivo. When practiced in vitro, any sterile, non-toxic routeof administration may be used. When practiced in vivo, administration ofthe compounds discussed above may be achieved advantageously bysubcutaneous, intravenous, intramuscular, intraocular, oral,transmucosal, or transdermal routes, such as, for example, by injectionor by means of a controlled release mechanism. Examples of controlledrelease mechanisms include polymers, gels, microspheres, liposomes,tablets, capsules, suppositories, pumps, syringes, ocular inserts,transdermal formulations, lotions, creams, transnasal sprays,hydrophilic gums, microcapsules, inhalants, and colloidal drug deliverysystems.

The immunogenic compositions are administered in a pharmaceuticallyacceptable form and in substantially non-toxic quantities. Inparticular, the immunogenic compositions can be administered in amountsappropriate to those individual compounds to produce an immune response.Appropriate doses can readily be determined by techniques well known tothose of ordinary skill in the art without undue experimentation. Such adetermination will be based, in part, on the tolerability and efficacyof a particular dose using techniques similar to those used to determineproper chemotherapeutic doses. Additionally, the compounds may beadministered in water with or without a surfactant.

Injectable preparations include sterile aqueous solutions or dispersionsand powders, which may be diluted or suspended in a sterile environmentprior to use. Carriers such as solvents or dispersion media containingwater, ethanol polyols, vegetable oils and the like may also be added tothe compositions described herein. Coatings such as lecithins andsurfactants may be used to maintain the proper fluidity of thecomposition. Isotonic agents such as sugars or sodium chloride may beadded, as well as products intended to delay absorption of the activecompounds, such as aluminum monostearate and gelatin. Sterile injectablesolutions are prepared according to methods well known to those of skillin the art and can be filtered prior to storage and/or use. Sterilepowders may be vacuum or freeze dried from a solution or suspension.Sustained-release preparations and formulations are also contemplated.Any material used in the compositions described herein should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. Antimicrobial compounds may optionally be added to thepreparations.

Although in some of the experiments that follow the compounds areadministered in a single dose, it should be understood that in aclinical setting, the compounds may be administered in multiple dosesover prolonged periods of time. In particular, the compounds may beadministered for periods up to about one week, and even for extendedperiods longer than one month or one year. In some instances,administration of the compounds may be discontinued and resumed at alater time.

All compound preparations may be provided in dosage unit forms foruniform dosage and ease of administration. Each dosage unit formcontains a predetermined quantity of active ingredient calculated toproduce a desired effect in association with a pharmaceuticallyacceptable carrier. Such a dosage would therefore define an effectiveamount of a particular compound.

A kit comprising the necessary components of an immunogenic compositionthat elicits an immune response to a selected immunogenic component arealso within the purview of the present invention.

Diagnosis

The invention also provides for a method of detecting Aap, AatP, AatA,AatB, AatC, and/or AatD in a sample which includes contacting a samplefrom a subject with an antibody to one or more of these proteins anddetecting binding of the antibody to the specific protein for which theantibody recognizes. Antibody binding is indicative of the presence ofthe specific protein in the sample. In one embodiment of the instantinvention, the presence of Aap, AatP, AatA, AatB, AatC, and/or AatD inthe sample is indicative of infection by EAEC. Since Aap and Aat arehighly specific for EAEC, the detection of these proteins on bacteria ina clinical sample suggests that the patient is infected with EAECbacteria.

The term “sample” includes material derived from an animal (e.g., fish,bird, mammal, human, etc.). Such samples include, but are not limitedto, hair, skin, tissue, cultured cells, cultured cell media, andbiological fluids. The term “tissue” refers to a mass of connected cells(e.g., CNS tissue, neural tissue, or eye tissue) derived from a human orother animal and includes the connecting material and the liquidmaterial in association with the cells. As used herein, the term“biological fluid” refers to liquid material derived from a human orother animal. Such biological fluids include, but are not limited to,blood, plasma, urine, semen, excrement, serum, serum derivatives, bile,phlegm, saliva, sweat, amniotic fluid, pleural fluid, and cerebrospinalfluid (CSF), such as lumbar or ventricular CSF.

The term “sample” also includes solutions containing the isolated Aap,AatP, AatA, AatB, AatC, and/or AatD polypeptides, media into which theseproteins has been secreted, and media containing host cells whichproduce these proteins. For example, a sample may be a protein samplewhich is to be resolved by SDS-PAGE and transferred to nitrocellulosefor Western blot analysis. The quantity of sample required to obtain areaction may be readily determined by one skilled in the art by standardlaboratory techniques. The optimal quantity of a sample may bedetermined by serial dilution.

A kit for diagnosing or determining the presence of EAEC in an animal isan embodiment within the scope of this invention. In one embodiment ofthis invention, a kit contains one or more antibodies (e.g., that havebeen described above) that recognize Aap, AatP, AatA, AatB, AatC, and/orAatD or a fragment of one or more of these proteins. The kit is usefulfor the detection of Aap, AatP, AatA, AatB, AatC, and/or AatD and has acompartmentalized carrier capable of receiving in a close confinement acontainer containing an antibody which binds to Aap, AatP, AatA, AatB,AatC, and/or AatD. As used herein, “a container” includes vials, tubes,and the like, each of the containers containing one of the separateelements to be used. In a preferred embodiment, the antibody which bindsto Aap, AatP, AatA, AatB, AatC, and/or AatD is detectably labeled. In aneven more preferred embodiment, the label is a radioisotope, abioluminescent compound, a chemiluminescent compound, a fluorescentcompound, a metal chelate, or an enzyme. The kit may alternativelycontain an antibody which recognizes an anti-Aap antibody, or anti-AatPantibody, or anti-AatA antibody, or anti-AatB antibody, or anti-AatCantibody, or anti-AatD antibody so that the kit can be used to determineif antibodies to one or more of these proteins are present in a sampleor tissue.

In a kit format, the antibody would be commercially bound to latexbeads. The beads would be mixed with a suspension of E. coli bacteria insaline. The suspension is rocked at room temperature for one minute andthe presence of agglutination of the suspension is read as a positivetest.

In yet another embodiment, the kit is useful for the detection of an aappolynucleotide and/or polynucleotides for one or more genes of the aatgene cluster. The kit is a compartmentalized carrier to receive in closeconfinement a container containing the nucleic acid probe thathybridizes to a polynucleotide for aap, and/or one or more genes of theatt gene cluster. Preferably, the nucleic acid probe that hybridizes tothe polynucleotide from the sample is detectably labeled. It ispreferred that the label is a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, oran enzyme.

Prevalence of the aat Cluster Among EAEC

The sequence of aatA is remarkably conserved between AAF/1-encodingstrain 17-2 and AAF/II-encoding strain 042. They are 96% identical atthe nucleotide level and 95% at the amino acid level. To assess theconservation of the aat cluster among clinical EAEC isolates, 31 strainsisolated from children of several districts around the world for aatAand aatBCD (also referred to as aatB, aatC, aatD) are examined by PCR.FIG. 4 shows the representative PCR results. In addition, the presenceof aggR is investigated by PCR to compare the prevalence of aat andaggR. The prevalence of aatA, aatBCD, and aggR in 31 EAEC isolates isshown below in Table 1 set forth below. The incidences of aatA andaatBCD are 71.0% (22/31) and 74.2% (23/31) respectively. It is notedthat aatA is always accompanied by aatBCD, except in one strain. Thisobservation suggests that the aat cluster is highly conserved among EAECclinical isolates. Nineteen of twenty-two aat-positive strains areaggR-positive, while five of nine aat-negative strains areaggR-negative. The presence of aggR significantly correlates with thatof the aat cluster (p=0.016). TABLE 1 Prevalences of the aat cluster andaggR in EAEC isolates. No. Strain aatA aatBCD aggR 1 042 (Peru) + + + 217-2 (Chile) + + + 3 Brazil 236 + + + 4 Mexico 60A + + + 5 Peru11145-1 + + + 6 Peru 11194-2 + + + 7 Peru 11232-1 + + + 8 Peru1132-1 + + − 9 Peru 1146-2 + + − 10 Peru 1177-1 + + + 11 Peru1192-1 + + + 12 Peru 133 + + + 13 Phil DS244-R3 + + + 14 PhilDS61-R2 + + − 15 Phil DS67-R2 + + + 16 Thai 103-1-1 + + + 17 Thai144-1-1 + + + 18 Thai 199-1-4 + + + 19 Thai 253-1-1 + + + 20 Thai309-1-1 + + + 21 Thai 44-1-1 + + + 22 Thai 6-1-1 + + + 23 Peru 11223-1− + + 24 Peru 1111-1 − − + 25 Peru 11191-1 − − + 26 Peru 1172-2 − − − 27Phil DS65-R3 − − − 28 Thai 435-1-1 − − − 29 Thai 501-1-1 − − + 30 Japan101-1 − − − 31 Serbia − − −

The Transcription of the aat Cluster and its Dependency on AggR

Initially, aatA, aatC, and aatD mutants of strain 042 are constructed.Each gene is then activated by the integration of a suicide plasmid,pJP5603. Each transcript is observed in the wild type but not in themutant (data not shown).

To assess the transcriptional linkage among the five genes, RT-PCR isperformed using several combinations of primers to the genes in the aatcluster (shown in Table 2). The RT-PCR assay using the primer aatPint-Fand aatAint-R yields a predicted size of product, but the assays usingthe combinations of primers derived from aatA, aatB, aatC, and aatDgenes do not yield products (data not shown). These results suggest thatthe aatP and aatA genes are transcribed polycistronically. The data alsoindicates transcriptional linkage of the other genes in the aat cluster.TABLE 2 Sequences of primers NAME GENE SITE SEQUENCE (5′ to 3′) RE SEQID NO: PCR aatA-F aatA 2723-2744 acggatccatgttaccagatataaatatag BamHISEQ ID NO: 18 aatA-R aatA 3766-3787 acgaattccatttcccctgtattggaaatg EcoRISEQ ID NO: 19 aatAint-F aatA 2893-2910 actctagatgaaatgcttagtgagag XbaISEQ ID NO: 20 aatAint-R aatA 3395-3412 acgaattcgatacccagactagcact EcoRISEQ ID NO: 21 aatCint-F aatC 4610-4630 actctagaagttggaaagacttcactgc XbaISEQ ID NO: 22 aatCint-R aatC 4889-4909 acgaattccggagagaaatgatacattaEcoRI SEQ ID NO: 23 aatDint-F aatD 5361-5380actctagaagttcttatgggttacttgg XbaI SEQ ID NO: 24 aatDint-R aatD 5841-5860acgaattcatcccatatttgtagtggag EcoRI SEQ ID NO: 25 aatW-F aat cluster001-022 acggatccggagacgtttggaggtgtatggg BamHI SEQ ID NO: 26 aatW-R aatcluster 6446-6465 acgcggccgctagcgttattgttcaacgcc NotI SEQ ID NO: 27aat-PA-R aatP and aatA 3864-3886 acgcggccgcacattaccttcaatcatgtcctc NotISEQ ID NO: 28 RT-PCR CAT-f cat tcactggatataccaccgtt SEQ ID NO: 29 CAT-Rcat ccactcatcgcagtactgtt SEQ ID NO: 30 aatA-F aatA 2723-2744atgttaccagatataaatatag SEQ ID NO: 31 aatA-R aatA 3766-3787catttcccctgtattggaaatg SEQ ID NO: 32 aatB-F aatB 3687-3709atgaaacagaaaatgaatttcag SEQ ID NO: 33 aatB-R aatB 4483-4508ctaatcatctattataatctcaaacg SEQ ID NO: 34 aatC-F aatC 4501-4523atgattagagtaaaaatacataa SEQ ID NO: 35 aatC-R aatC 5108-5130ctatgtatttaatagttggatta SEQ ID NO: 36 aatD-F aatD 5142-5166atgaaattcgctattgtcttattgt SEQ ID NO: 37 aatD-R aatD 6328-6356tcatatctgtgtaaataaaaaaggttccg SEQ ID NO: 38 aatPint-F aatP 2004-2023ctcgataacagagtcaatgc SEQ ID NO: 39 aaP-F aatP 1432-1457ctttgcactattatctaaatgaggcg SEQ ID NO: 40 aatP-R aatP 2522-2548atctttccttttattgcattaacaggg SEQ ID NO: 41*Restriction Enzyme shown as the underlined sequence.

To test the effect of AggR on the transcription of the aat cluster,RT-PCR for the aatA transcript in strains 042 and 042aggR is performed.A product of the predicted size is seen in 042 but is absent in 042aggR. (See FIG. 6). To confirm that AggR is required for aat clustertranscription, the transcription of aatA in the complement strain isfound to be under control of the arabinose-dependent promoter,042aggR(pBADaggR). (See FIG. 6, lanes 4 and 5). The aatA transcriptionis restored in 042aggR(pBADaggR) when cells were grown in the presenceof arabinose (ara-inducing conditions) but not in the presence ofglucose (repressing conditions). aat transcription was not observed inthe negative control strain 042aggR(pBAD30). (See FIG. 6, lane 3). Usingsimilar methods, AggR is also shown to be essential in the transcriptionof aatP, aatC, and aatD (data not shown).

Localization of the AatA Protein

An AatA-His fusion protein is expressed and purified (see below). Thisprotein is then used to generate a highly specific AatA antiserum.AatA-specific antibodies are concentrated by affinity purificationagainst the purified protein (see below). Western immunoblots are thenprepared from whole cell, periplasm, and outer membrane. A band of theexpected size is observed in whole cell lysates of 042 cultured inDulbecco's minimum essential medium (DMEM) supplemented with 0.45%glucose (high-glucose DMEM) but not in 042aatA. (Data not shown). Thesame size of band is observed in the outer membrane, but not in theperiplasm fraction. AatA is not observed in the culture media (data notshown). Thus, AatA exists mainly on the outer membrane. To determinewhether AatA is translocated to the outer membrane using the energygenerated by the ABC transporter junction of AatC, 042aatC and 042aatDare examined for AatA by Western immunoblot. AatA is observed in theouter membranes of both 042aatC and 042aatD (data not shown).

The aat Cluster is Associated with the Secretion of a Dispersin Aap

The transmembrane domain of the importers among the prokaryote ABCtransporters invariably carries a conserved motif, EAA, in theC-terminal. (Holland, J Mol Biol 293:381-399 (1999)). Because the motifis not observed in the amino acid sequences of the aat cluster, it ishypothesized that the aat cluster exports an unidentified protein acrossthe inner and/or outer membrane. To verify the hypothesis, thedifferences in secreted proteins between the wild type and the mutant insodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ofculture media precipitated with trichloroacetic acid (TCA) areinvestigated. Strain 042 and 042aatA was cultured in high-glucose DMEMover night. The culture media were precipitated with TCA, boiled for 5min, and separated using 15% SDS-PAGE. No significant differences ofSDS-PAGE patterns of the culture media of L-broth are observed. However,a slight difference in high-glucose DMEM (FIG. 4A), in which AggR isknown to be expressed, is observed.

Silver staining of the gel shows a band of 10 kD in wild type EAEC, butnot in 042aatA. Because the molecular size of the mature Aap is known tobe 10.2 kD, this secreted protein is predicted to be Aap. Aap is knownto bind non-covalently to the bacterial cell surface, and to be easilyreleased into the culture media by adding Triton X-100 to a finalconcentration of 0.1% into the medium. SDS-PAGE of the supernatant ofthe culture is performed with 0.1% Triton X-100. (See FIG. 4B). SDS-PAGEof the culture media of 042, 042aatA, 042aatC, 042aatD, and 042pet. Thestrains were cultured in high-glucose DMEM containing 0.1% Triton X-100for 6 hours. The culture media were precipitated with TCA, boiled for 5min, separated using 15% SDS-PAGE, and stained with Coomassie blue. Theband in the wild type is easily observed by the Coomassie-staining. Onthe other hand, the band is not observed in 042aatA, 042aatC, and042aatD. To eliminate the possibility of an effect of pJP5603, themutant 042pet, which harbors pJP5603 integrated into the gene of theEAEC toxin pet, is examined as a control. The 042pet shows the same bandas the wild type.

This 10 kDa secreted protein is confirmed to be Aap by westernimmunoblot. (See FIG. 5). Each strain was cultured in high-glucose DMEMfor 6 hours with and without 0.1% Triton, and the supernatant wasprecipitated with TCA. The samples were then boiled and separated using15% SDS-PAGE. The western immunoblot was performed by standard methodsusing the specific polyclonal antibodies against Aap.

As shown in FIG. 5, Aap is observed in the supernatant of wild type EAECin high-glucose DMEM, but not in 042aatA. The amount of secreted Aapincreases after the addition of 0.1% Triton X-100 in both the wild typeand 042aatA. However, the level of secreted of Aap in the wild type ismuch higher than that in 042aatA. The level of Aap remaining in the cellpellet is higher in 042aatA than in the wild type.

The level of secreted Aap is also reduced in 042aatC and 042aatD, butnot in 042pet. (See FIG. 6A). Western Immunoblot analysis was conductedfor 042, 042aatA, 042aatC, 042aatD, and 042pet in the supernatant andpellet. Each strain was cultured in high-glucose DMEM with 0.1% TritonX-100 for 6 hours. The supernatant was then precipitated with TCA,boiled, and separated using 15% SDS-PAGE. Western Immunoblot wasperformed by standard methods using the specific polyclonal antibodyagainst Aap. The degree of reduction is estimated to be less than10-fold.

In addition, western immunoblot analysis was conducted for Aap in theperiplasms of 042, 042aatA, 042aatC, and 042aatD. Each strain wascultured in high-glucose DMEM over night. The periplasms were extractedas described below in the section entitled “Preparation and Analysis ofCellular Fractions”.

Aap levels in the pellet and the periplasm in 042aatA, 042aatC and042aatD are higher than in the wild type, suggesting that unsecreted Aapremains inside of the outer membrane, presumably in the periplasm. (SeeFIGS. 6A and 6B).

To verify that aatA is associated with the secretion of Aap, 042aatA iscomplemented. Initially, pJNAD (see FIG. 3), which carries the 4.5-kbfragment encoding aatABCD cloned into the single copy expression vectorpZC320, is introduced into 042aatA. However, this complement 042aatA(pJNAD) does not restore Aap secretion completely (data not shown).RT-PCR assay shows that the transcriptional level of aatA in 042aatA(pJNAD) is very low compared to the wild type (data not shown).Therefore, it is believed that the promoter of aatA exists in theupstream region of aatP.

pJNW, which contains the 6.5-kb whole aat cluster including the upstreamregion of aatP cloned into the pZC320 (see FIG. 3), is introduced into042aatA. This complement is defined as 042aatA(pJNW). AatA is detectedin the whole cells and the outer membrane of 042aatA(pJNW) by WesternImmonoblot. (See FIG. 7A). Western immunoblot analysis was conducted forAatA in the whole cells, the outer membrane, and the supernatant of 042,042aatA, and 042aatA (pJNW). Each strain was cultured in high-glucoseDMEM over night. The outer membrane fractions were prepared as describedin the section below entitled “Preparation and Analysis of CellularFractions”. The outer membrane fraction and whole cell samples wereseparated using 10% SDS-PAGE after being boiled. The supernatant wasprecipitated with TCA, boiled, and separated using 15% SDS-PAGE. WesternImmunoblot was performed by standard methods using the specificpolyclonal antibodies against AatA. The secretion level of Aap isrestored in 042aatA(pJNW) to a level that is almost the same as the wildtype. (See FIG. 7B).

The complement 042aatC(pJNW) and 042aatD(pJNW) also restores Aapsecretion. (See FIG. 8). The results illustrated in FIG. 8 are theresults of a western immunoblot analysis of Aap in the supernatant of042, 042aatC, 042aatC(pJNW), 042aatD, and 042aatD(pJNW). In particular,each strain was cultured in high-glucose DMEM for 6 hours with 0.1%Triton. The supernatant was precipitated with TCA, boiled, and separatedusing 12.5% SDS-PAGE. Western immunoblot was performed by standardmethods using the specific polyclonal antibodies against the Aapprotein;

To determine the possibility of translational polar effects, pJNPA, a3.9-kb fragment encoding aatPA cloned into pZC320 (FIG. 3), isintroduced into 042aatA. This complement, 042aatA(pJNPA), restores thesecretion of Aap to almost the same level as 042aatA(pJNW), suggestingthat there is no polar effect of the mutagenesis.

Experimental Procedures

Bacterial Strains, Plasmids, and Growth Conditions

Strains and plasmids used herein are listed in Table 3 set forth below.TABLE 3 Bacterial strains and plasmids Strain or plasmid CharacteristicsReference or source Strains 042 Wild-type EAEC prototype strain Nataro1985 042aatA 042 harboring pJP5603 integrated into the aatA gene. Thiswork Km^(R) 042aatC 042 harboring pJP5603 integrated into the aatC gene.This work Km^(R) 042aatD 042 harboring pJP5603 integrated into the aatDgene, This work Km^(R) 042aatA(pJNW) 042aatA carrying the whole aatcluster cloned into This work pZC320. Km^(R), Ap^(R) 042aatC(pJNW)042aatC carrying the whole aat cluster cloned into This work pZC320.Km^(R), Ap^(R) 042aatD(pJNW) 042aatD carrying the whole aat clustercloned into This work pZC320. Km^(R), Ap^(R) 042aatA(pJNAD) 042aatAcarrying aatA, aatB, aatC, and aatD cloned This work into pZC320.Km^(R), Ap^(R) 042aatA(pJNPA) 042aatA carrying the aatP and aatA genecloned into This work pZC320. Km^(R), Ap^(R) DH5α λpir K12 E. colilysogenized for the pir gene, which Elliott 1997 permits replication ofR6K plasmid replicons S17-1 λpir Conjugative K12 lysogenized for pir.Tet^(R), Km^(R) Simon 1983 042pet 042 harboring pJP5603 integrated intothe pet gene, Henderson 1999 Km^(R) 042aggR 042 carrying TnphoA insertedinto the aggR gene Sheikh 2001 042aggR(pBADaggR) 042aggR carrying theaggR gene cloned into Sheikh 2002 pBAD30 to permit expression of AggR inthe presence of arabinose 042aggR(pBAD30) 042aggR carrying pBAD30 usedas a background for This work 042aggR(pBADaggR) Plasmids pET21a(+) T7promoter-driven expression vector, Ap^(R) Novagen pJP5603 3.1-kb R6Ksuicide plasmid, Km^(R) Penfold 1992 pZC320 7.5-kb single copy vector,Ap^(R) Shi 1995 pAatA 1065-bp fragment of the aatA gene cloned into Thiswork multiple cloning site of pET21a(+) to provide IPTG- inducibleexpression of the AatA protein as a 6-His fusion, Ap^(R) pINTA 520-bpinternal fragment of the aatA gene in This work pJP5603 pINTC 300-bpinternal fragment of the aatC gene in This work pJP5603 pINTD 500-bpinternal fragment of the aatD gene in This work pJP5603 pJNAD 4.5-kbPCR-derived fragment encoding aatA, aatB, This work aatC, and aatD. pJNW6.5-kb PCR-derived fragment of the whole aat This work cluster clonedinto pZC320 pJNPA 3.9-kb fragment encoding aatP and aatA cloned intoThis work pZC320 pBAD30 High copy number expression vector permittingGuzman expression of foreign genes under control of the arabinose operonpromoter pBADaggR aggR cloned into multiple cloning site of pBAD30 toSheikh 2002 permit expression of AggR in the presence of arabinose

Strain 042 was isolated from a child with diarrhea in the course of anepidemiological study in Lima, Peru, in 1983 (Nataro et al., J InfectDis 152:560-565 (1985)). This strain has also been shown to causediarrhea in adult volunteers (Nataro et al., J Infect Dis 171:465-468(1995)). EAEC strains used in retrospective PCR analysis are from theCenter for Vaccine Development and were isolated during epidemiologicalstudies in various sites throughout the world. All strains are stored at−70° C. in Trypticase soy broth with 15% glycerol. All E. coli strainsare grown aerobically at 37° C. in Luria-Bertani (LB) medium unlessotherwise stated. Antibiotics are added at the following concentrationswhere appropriate: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml; andnalidixic acid, 50 μg/ml.

Molecular Cloning and Sequencing Procedure

Plasmid DNA purification, restriction, ligation, transformation, andagarose gel electrophoresis are performed by standard methods (Sambrooket al., Molecular cloning: a laboratory manual, 3rd ed. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)). Plasmid DNA,except pAA2, as extracted using the QIAprep Spin Miniprep Kit or theQIAEX II Gel Extraction kit (Qiagen, Valencia, Calif.). The extractionof DNA fragments from agarose gels is performed using the Concert RapidPCR purification kit (Life Technologies, Rockville, Md.). Plasmid DNA isintroduced into E. coli DH5α, DH5α λpir, and S17-1 λpir by heat shocktransformation of competent cells according to the method of Hanahan (J.Mol. Biol. 166:557-580 (1983)) or into 042aatA, 042aatC, and 042aatD byelectroporation using a Gene Pulser II system (Bio-rad, Hercules,Calif.). DNA sequence analysis is performed at the University MarylandDepartment of Microbiology & Immunology Biopolymer Facility on anApplied Biosystems model 373A sequencer; template DNA was purified byusing minicolumns from Amersham-Pharmacia-Biotech (Piscataway, N.J.).

PCR Procedure

Amplifications are performed with 500 ng of purified genomic DNA astemplates in a 50 μl reaction mixture containing 2.5 U of Taq DNApolymerase, 0.5 μM each primer, 0.2 mM each deoxynucleosidetriphosphate, 2 mM MgCl₂ and 5 μl of the manufacturer's buffer(Invitrogen, Carlsbad, Calif.). Amplification reactions are performed inan MJ Minicycler for 5 minutes at 94° C., followed by 30 cycles at 94°C. for 30 seconds, 50° C. for 40 seconds, and 72° C. for 1 minute perkb, concluding with extension at 72° C. for 10 minutes unless otherwisestated. The products are separated by 0.7˜1.0% agarose gels, stainedwith ethidium bromide, and visualized with UV transillumination. Theprimers sequences used in this study are shown in Table 2 above.

RT-PCR Analysis

Total RNA is extracted with an RNeasy mini kit (Qiagen Inc., Valencia,Calif.) from LB culture shaking to mid-log phase. Preparations aretreated with RNase-free Dnase I (Roche Molecular Biochemicals,Indianapolis, Ind.) to eliminate contaminating DNA. The absence ofcontaminating genomic DNA in RNA preparations is verified by performingPCR for the chromosomal chloramphenicol acetyltransferase (cat) gene. Tosynthesize cDNA, total RNA (2 μg) is subjected to reverse transcriptase(RT) reactions using Thermoscript RT (Invitrogen) and gene-specificreverse primers according to the manufacturer's instructions. Primersused for RT-PCR are shown in Table 2. Amplification reactions wereperformed in an MJ Minicycler for 5 minutes at 94° C., followed by 30cycles of 94° C. for 30 seconds, 50° C. for 40 seconds, and 72° C. for 1minute per kb, concluding with extension at 72° C. for 10 minutes unlessotherwise stated. The products were separated by 0.7˜1.0% agarose gels,stained with ethidium bromide, and visualized with UVtransillumination.

Isolation of Aap

Although an isolation technique for isolating Aap is described below,the isolation of AatP, AatA, AatB, AatC, and AatD is conducted in a likemanner.

Given that Aap is the dominant protein non-covalently attached to thesurface of EAEC bacteria, it can easily be isolated. The bacteria arecultured overnight at 37° C. in Minimal Essential Medium or L-broth,either containing 0.05% glucose to maximize Aap expression. Aftercultivation, the bacteria are pelleted by centrifugation for 20 minutesat 20,000×g. The bacterial pellet is then resuspended in PBS with 0.1%Triton and incubated at 37° C. for one hour. The bacteria are thenpelleted as before. The supernatant contains substantially pure Aapprotein (ca. 95%).

AatA Expression and Purification

Although the AatA expression and purification are described below, theexpression and purification of Aap, AatP, AatB, AatC, and AatD isconducted in the same manner.

The aatA gene product is expressed by cloning a 1064-bp fragmentgenerated by PCR into the BamHI and EcoRI sites of the expression vectorpET21a(+) (Novagen, Madison, Wis.). The primer sequences of aatA-F and-R for amplification are shown in Table 2. The protein is therebysynthesized as a fusion with a hexa-His-tag to the carboxy terminus andT7-tag. Protein expression is achieved by incubating an LB culture ofthe construct at 37° C. with shaking until an optical density at 600 nmof 0.5 to 0.6 was reached. Cells were induced with a final concentrationof 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma ChemicalCo., St. Louis, Mo.) for 3 hours. The fusion protein is purified in thedenaturing condition by passage through a metal affinity matrixaccording to the standard protocols supplied with the Talon metalaffinity resin (Clontech, Palo Alto, Calif.). The column eluate isdialyzed overnight against PBS containing 8 M urea (pH 7.4). His-tagfusion protein is separated by SDS-PAGE and detected by staining withCoomassie brilliant blue R250. To determine the identities of theprotein of interest, the bands are excised from gel and analyzed by massspectrometry at the Protein and Nucleic Acid Research Facility, StanfordUniversity School of Medicine, Palo Alto, Calif.

Preparation of AatA-Specific Antibodies

Although the preparation of AatA-specific antibodies are describedbelow, antibodies to Aap, AatP, AatB, AatC, and AatD are prepared in thesame manner.

Antibodies Rabbit antiserum specific for AatA is raised by subcutaneousinjection of AatA preparations in Freund's adjuvant as described inHarlow et al., 1998. Affinity purification of antiserum is performed topurify AatA-specific antibodies. His-tag AatA fusion proteins (10 mg) iscoupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden).Rabbit antiserum is adsorbed by mixing with His-AatA protein-coupledSepharose for 2 hours at room temperature. After washing, boundantibodies are eluted with 0.2 M glycin (pH 1.85) and immediatelyneutralized with 1 M Tris-HCl (pH 8.5). The eluate is dialyzed againstPBS, concentrated using a Vivaspin 20, 50,000 MWCO (Vivascience,Hannover, Germany), and then subsequently is used for WesternImmunoblot.

Monoclonal antibodies could be raised against Aap by injecting thepurified protein into mice. 50 mcg of purified Aap protein would beinjected with Freund's adjuvant into each of three mice on days 0, day14, day 28 and day 43. Three days after the final boost, mouselymphocytes are isolated and fused with myeloma cells according tostandard procedures. Twenty four days after fusion, cell lines arescreened for antibody production by examining the medium of individualcolonies for the presence of antibodies to Aap by Western blot. Colonieswith high level specific antibody production will be expanded to fullcell culture flasks and saved for future use.

Polyclonal antibodies are raised by injection of gel slices into NewZealand White rabbits. A preparation containing 20 mcg of pure Aapprotein is added to SDS-PAGE running buffer (available commercially fromBio-Rad) and separated on an SDS-PAGE gel (15% gel) for 4 hours. The gelis then stained with Coomassie Brilliant Blue (Bio-Rad) and visualized.The band corresponding to Aap is easily identified by size. The band isexcised from the gel using a razor blade, and the band is chopped intovery small slices with the blade. The slices are mixed with an equalvolume of PBS and drawn in and out through a 20 gauge hypodermic needleto further homogenize the gel slice in the PBS. When only small (ca. 1mm) lumps are left, the slurry is injected subcutaneously into thesubcutaneous region on the back of a 1.5 kg New Zealand White rabbit.Typically the injectate is divided into 3-4 injections. This procedureis repeated two weeks later, then again four weeks after the secondinjection. One week after the third injection, the rabbit is bled forantiserum. The blood is allowed to clot at room temperature for 3 hr,then the blood is centrifuged at 15,000=g for 10 minutes. Thesupernatant is removed and stored at −20° C.

Preparation and Analysis of Cellular Fractions

To prepare culture supernatant fractions, strains are grown overnight orfor 6 hours at 37° C. in 5 ml of high-glucose DMEM. The growth rate ofmutants is almost the same as that of the wild type (data not shown).After centrifugation at 13,000×g for 5 minutes, proteins in thesupernatant are precipitated with trichloroacetic acid (TCA). One-fourthvolume of TCA containing 0.4% (wt/vol) deoxycholate is added to thesupernatant in an eppendorf tube, and incubated on ice for 30 minutesafter vortexing. The pellet is centrifuged at 14,000×g for 15 minutesand washed with 1.5 ml of acetone for 15 minutes at room temperature.The pellet is again collected by centrifugation at 14,000×g for 15minutes, dried, and suspended in 50 μl of Laemmli sample buffer. Todetect Aap that attached to the outer membrane non-covalently, strainsare grown in medium with 0.1% Triton X-100. The supernatant isprecipitated with TCA.

Outer membrane proteins are extracted from cultures grown overnight at37° C. in 20 ml of high-glucose DMEM. Bacteria are harvested bycentrifugation at 6,000×g for 10 minutes at 4° C. and resuspended in 3.0ml of 10 mM Tris, pH 8.0. The cells are lysed with a French press andcentrifuged for 30 minutes at 13,000 g at 4° C. The pellet isresuspended in 240 μl of 10 mM Tris, pH 8.0, 60 μl of 10% Triton X-100,and 1.5 μl of 1M MgCl₂ (Skare, et al., J Bacteriol 178:4909-4918 (1996))or 35 μl of distilled H₂O and 265 μl of Sarkosyl (Amako, et al.,Microbiol Immunol 40:749-754 (1996)). It is incubated at roomtemperature for 20 minutes, and the pellet is resuspended in 50 μl ofLaemmli sample buffer.

Periplasms are extracted from cultures grown overnight at 37° C. in 5 mlof high-glucose DMEM. Bacteria are harvested by centrifugation at3,800×g for 10 minutes and resuspended in 500 μl of lysis buffer (50 mMTris, pH 8.0, 3 mM EDTA, and 01% Triton X-100) (Thorstenson, et. al., JBacteriol 179:5333-5339 (1997)). The suspension is kept on ice for 30minutes, and centrifuged for 15 minutes at 3,800×g. The supernatant isstocked and the pellet is resuspended in 500 μl of lysis buffer. Thesuspension is centrifuged for 15 minutes at 3,800×g. The supernatantsare collected and precipitated with TCA as described above. The pelletis resuspended in 100 μl of Laemmli sample buffer.

One-dimensional SDS-PAGE (Laemmli et al., J Mol. Biol. Vol. 47(1):69-85(1970)) is performed using 10˜15% (wt/vol) acrylamide separating gelsand 4.0% (wt/vol) acrylamide stacking gels. Samples are routinely heatedfor 5 minutes at 100° C. in Laemmli sample buffer (Laemmli et al.,Nature 227:680-685 (1970)) prior to loading. Proteins are detected bystaining with Coomassie brilliant blue or Silver Stain Kit (Bio-RadLaboratories, Inc., Hercules, Calif.).

Western Immunoblot Analysis

Preparative protein samples were boiled for 5 minutes, separated bySDS-PAGE (10.5%, 12.5%, or 15%), and transferred onto Immobion-Pmembranes (Millipore). Dried skimmed milk (5% [wt/vol]) is used as ablocking reagent. For detection of AatA and Aap, an anti-AatA specificantibody and an anti-Aap antiserum are used at a dilution of 1:1,000 and1:8,000, respectively. Antigen-antibody complexes are reacted with ahorseradish peroxidase-conjugated goat anti-rabbit IgG at a dilution of1:40,000 and visualized using a chemiluminescence ECL kit (Amersham,Uppsala, Sweden).

Mutagenesis and Complementation

To construct the aatA mutant, an internal portion of the aatA gene(nucleotides 2893 to 3412) is generated by PCR and cloned into XbaI andEcoRI sites of the suicide vector pJP5603, whose replication requires acopy of the R6K pir-encoded π protein supplied in trans (Penfold et al.,Gene 118:145-146 (1992)). The sequences of primers used to amplify theinternal fragment are shown in Table 2. The resulting plasmid, pINTA, ispropagated in E. coli DH5α λpir prior to transformation into the donorE. coli strains in S17-1 λpir. The mutant strain is then obtained byconjugal mating between the wild type parent strain 042 (which isnalidixic acid resistant) and E. coli strains in S17-1 λpir.Transconjugants are selected on LB agar supplemented with kanamycin andnalidixic acid. This process results in integration of pINTA into thehomologous site in the aatA gene (and hence a merodiploid state). Theresulting strain is designated 042aatA. The insertion of the suicideplasmid into the native aatA gene is confirmed by restriction analysisof the pAA2 plasmid using MluI and BamHI. Lack of the aatA transcriptand the AatA protein is confirmed by RT-PCR and Western immunoblot,respectively.

To construct the aatC and the aatD mutant (042aatC and 042aatD,respectively), an internal portion of each gene (nucleotides 4610 to4909 and 5361 to 5860, respectively) is generated by PCR and cloned intopJP5603 (pINTC and pINTD, respectively). The sequences of primers usedto amplify the internal fragment are shown in Table 2. The 042aatC andthe 042aatD are generated as described above. Lack of the aatC and aatDtranscript is confirmed by RT-PCR.

To construct the trans complement of mutants, the whole aat cluster isamplified by PCR and cloned into the single copy vector pZC320. Theprimer sequences of aatW-F and -R used for amplification are shown inTable 2. Amplifications are performed with 500 ng of purified genomicDNA as templates in a 50 μl reaction mixture containing 2.5 U ofPlatinum pfx DNA polymerase, 0.5 μM each primer, 0.3 mM eachdeoxynucleoside triphosphate, 2 mM MgCl₂ and 5 μl of the manufacturer'sbuffer (Invitrogen, Carlsbad, Calif.). Amplification reactions areperformed in an MJ Minicycler for 5 minutes at 94° C., followed by 30cycles at 94° C. for 30 seconds, 54° C. for 40 seconds, and 68° C. for6.5 minutes, concluding with extension at 68° C. for 10 minutes. The6.5-kb PCR product is cloned into the BamHI and NotI sites of the vectorpZC320. This plasmid is designated pJNW. pJNW is introduced into042aatA, 042aatC, and 042aatD by electroporation.

pJNPA, 3.9-kb fragment encoding aatP and aatA cloned into pZC320, isconstructed in the same manner as described above using the primeraatW-F and aatPW-R. pJNPA is introduced into 042aatA by electroporation.pJNAD, the 4.5-kb fragment encoding aatABCD cloned into pZC320, is alsoconstructed, and introduced into 042aatA.

Computer and Statistical Analysis

Analysis of DNA and protein sequence is performed using programsavailable through the National Center for Biotechnology Information(www.ncbi.nlm.nih.gov) and the ExPASy server of the Swiss Institute ofBioinformatics (www.expasy.ch). Statistical testing is performed usingFisher's exact test. Probability values less than 0.05 are consideredsignificant.

Identification of the aap Gene

As mentioned above, aap is located on pAA approximately 843 nucleotidesupstream from the aggR start codon. SDS-PAGE was performed by thestandard method of Laemmli et al., J Mol Biol., Vol. 47(1):69-85 (1970)as described in Ausubel et al. All of the gels are run on theMini-Protean III electrophoresis system from Bio-Rad using 1 mm spacers.All of the reagents for SDS-PAGE are purchased from Bio-Rad, Inc.(Hercules, Calif.). The gels used in the SDS-PAGE are 10% polyacrylamideunless otherwise specified. All of the samples are prepared in BioRadSDS-PAGE running buffer and boiled for 10 minutes prior to gel loading.The gels were stained in Coomassie blue using gel staining reagents fromBioRad according to manufacturer's protocols.

The aggR gene is located downstream of the fimbrial subunit (aafA) onthe pAA plasmid of prototype EAEC strain 042. This region is completelyconserved in the sequence of the AAF/I-encoding plasmid of strain 17-2,with 97% sequence homology and conservation of all open reading frames.

Further, it was determined that the Aap sequence featured two cysteineresidues that may form a disulfide loop, but there was no otherdistinguishing amino acid motifs or properties.

Construction of an aap Knockout Mutation in Strain 042

An aap mutant is constructed in E. coli strain 042 by a single crossoverinsertion of suicide plasmid pJP5603 using the following methods:

A DNA fragment internal to aap is synthesized by PCR using the followingprimers: (SEQ ID NO. 14) 5′-ATGGTACCTTGTTATCTTTTCTGGCATCTTGGGT-3′ (SEQID NO. 15) 5′-ATGAGCTCTGGAGGGGGTAACAACCCCTTTGAAGT-3′.PCR is performed using the Optiprime kit (buffer 8) from Stratagene,Inc. (La Jolla, Calif.) according to manufacturer's instructions.

The PCR reaction is performed in a PTC-150 Minicycler from MJ Research,Inc., (Watertown, Md.) using the following program: 40 cycles at 94° C.for 1 minute, 58° C. for 90 seconds, 72° C. for 2 minutes; followed by asingle extension reaction at 72° C. for 10 minutes.

Next, the reaction product is digested with restriction enzymes KpnI andSacI (Gibco/BRL, Gaithersburg, Md.) for 3 hours and then separated byagarose gel electrophoresis using standard methods. The fragment isexcised from the gel and cloned into suicide vector pJP5603 (obtainedfrom Dr. J M. Pemberton and referenced in Penfold R J and Pemberton J M,Gene 118:145; 1992), previously digested with the same restrictionenzymes for 3 hour. E. coli suicide vector pJP5603 is kanamycinresistant and has the suicide R6K replicon.

Ligation is performed by standard protocols disclosed in Ausubel et al.with a DNA ligase obtained from Gibco/BRL for 18 hours at roomtemperature using a buffer provided by the manufacturer and ATP obtainedfrom Gibco/BRL. The volume of the ligation reaction is 20 μl. 5 μl areused to transform E. coli DH5α(λpir) using standard calcium chloridetransformation conditions according to standard protocols. Therecombinant plasmid is then purified using the Qiagen midi-plasmid kitaccording to manufacturer's instructions. (Qiagen, Inc., Valencia,Calif.). 1 μg of purified plasmid DNA is transformed into strainS117-1(λpir) according to the standard calcium chloride technique.

Strain S17-1(λpir) is used for mobilization of the plasmid into strain042. S17-1(λpir) carrying the pJP5603aap construct and recipient strain042 (Nalidixic acid resistant) are each grown in Luria broth to logphase (3 hr) at 37° C. 100 μl of each broth culture are then mixed on acellulose nitrate disc (obtained from Scheicher and Schuell, Inc.,Keene, N.H.) which had been applied to a Luria agar plate withoutantibiotics. The plate is incubated overnight at 37° C. The followingday, bacterial growth is resuspended in L-broth and plated on L-agarwith ampicillin (100 μg/ml) and nalidixic acid (100 μg/ml). Afterovernight incubation, individual colonies are picked and the insertionof the suicide plasmid into the native aap gene is confirmed byextracting the 100 kb pAA2 plasmid and digesting it with BamHI andHindIII restriction enzymes. Since the pattern of digestion of thenative plasmid is known, the sites of insertions are be easilydetermined.

The resulting E. coli strain is designated 042aap. The constructcontained the pJP5603 plasmid integrated into the aap gene on nativeplasmid pAA2. The method of inactivation results in a merodiploidduplicate for the aap fragment cloned into pJP5603. Lack of Aapexpression was confirmed by Western blot as follows. 042aap is grown inL-broth for 3 hours at 37° C. with shaking. The bacteria are pelleted bycentrifugation (10,000×g) and 20 μl of the culture supernatant are mixedwith SDS-PAGE running buffer (obtained from Bio-Rad Inc., Hercules,Calif.), then separated on a 10% SDS-PAGE gel. The gel is thentransferred to nitrocellulose paper by electroblotting. Electroblottingis performed using a Bio-Rad Mini-Transfer system at 60 V for 4 hours at4° C. in Towbin's Tris/Glycine buffer with 20% methanol.

After transfer to nitrocellulose membrane (Schleicher and Schuell,Keene, N.H.), the blot is blocked in phosphate-buffered saline with 0.5%Tween 20 (Sigma Chemical Co., St. Louis Mo.) with 5% skim milk(Carnation, Nestle, Inc., Glendale, Calif.) overnight at 4° C. The blotis then incubated with anti-Aap antibody (raised as described below) ata dilution of 1:2000 in PBS 0.5% Tween-20 (Sigma Chemical Co., St. LouisMo.) for 18 hours. Next, the blot is incubated with 0.5% skim milk for 2hours at room temperature and then washed three times with PBScontaining 0.5% Tween-20. The blot is then reacted for 1 hour at roomtemperature with a secondary antibody, which was a 1:40,000 dilution (inPBS Tween) of goat anti-rabbit-horse radish peroxidase conjugate.(Amersham Pharmacia Biotech, Piscataway, N.J.). The blot is then washedthree times with PBS Tween. The blot is then developed with anchemiluminescence kit from Amersham Pharmacia according tomanufacturer's instructions. The blot is then air dried and exposed toX-ray film. (X-omat film, Kodak, Rochester, N.Y.).

All of the PCR reactions were performed using buffers from StratageneInc. (La Jolla, Calif.) and protocols from the manufacturer. Detailsspecific for each PCR reaction are provided below.

Isolation and Purification of Aap Protein and Generation of Antibodies

For purification of the Aap protein, the aap polynucleotide is ligatedinto plasmid pQE70 (with the resultant plasmid designated pAap)downstream of the lac promoter and upstream of six histidine residues. Anative Aap protein carrying an additional six histidine residues fusedto the carboxy terminus of the protein is produced. The aap gene isamplified by PCR using primers with the following sequences: (SEQ ID NO:16) 5′-ACATGCATGCAAAAAATTAAGTTTGTTATC-3′; and (SEQ ID NO: 17)5′-CGGGATCCAACCCATTCGGTTAGAGC-3′.

Amplification is performed using buffer number 8 and other reagents inthe Opti-Prime kit from Stratagene, Inc. (La Jolla, Calif.) according tomanufacturer's protocols. The PCR reaction is performed in an PTC-150Minicycler thermal cycler (MJ Research, Inc.) programmed for thefollowing sequence of steps: 94° C. for 3 minutes; followed by 35 cyclesat 94° C. for 1 minutes, 43° C. for 3 minutes, 72° C. for 2 minutes;followed by a final extension at 72° C. for 10 minutes. This reactionresults in a polynucleotide fragment with SphI and BamHI restrictionsites at the aap upstream and downstream ends respectively.

After amplification, the PCR product is digested with SphI and BamHI(according to manufacturer's instructions; Gibco/BRL, Gaithersburg, Md.)and ligated into expression vector pQE70, digested with the sameenzymes. The ligation reaction is used to transform E. coli strain DH5αby the calcium chloride technique. The resulting plasmid is designatedpAap.

Plasmid pAap DNA is isolated from DH5αpAap using the Qiagen plasmidextraction kit according to manufacturer's instructions (Qiagen,Valencia, Calif.). The plasmid DNA is confirmed to be the desiredconstruct by restriction digestion using SphI and BamHI. PCR isperformed using the same primers and conditions employed to generate thefragment used for cloning. The construct produces a product of thedesired size.

The pAap plasmid is extracted as described above then electroporatedinto E. coli 042aap. 042aap is made competent by the method described inAusubel et al., as follows: 042aap is grown overnight in 500 ml L-brothwith shaking to an OD600 of 0.6. The culture is then chilled on ice andharvested by centrifugation. The pellet is resuspended in 500 mlice-cold water and centrifuged again. This process is repeated. Thefinal pellet is resuspended in 5 ml ice water.

Electroporation is performed using a Gene Pulser from Bio-Rad, Inc. 0.5μg purified plasmid DNA resuspended in distilled water is added to 0.5ml cell suspension in a 0.2 cm cuvette from Bio-Rad. The Gene Pulser isset for 2.5 kV, 25 μF, 200 ohms and a single pulse is applied. 1 ml SOCmedium (Gibco/BRL, Gaithersburg, Md.) is then added, and the culture isincubated at 37° C. for 30 minutes with shaking prior to plating onL-agar plates with 100 mcg/ml ampicillin (Sigma).

Western immunoblot analysis of E. coli 042aap(pAap) using anti-Aapantibodies indicate the restoration of Aap expression. Levels of Aapexpression in E. coli 042aap(pAap) in the absence of IPTG induction areslightly higher than native levels.

The Aap-6His fusion protein is purified from a 100 ml culture volumegrown in L-broth with shaking at 37° C. until an OD of 0.6 is reached.At this point, IPTG (Sigma Chemical Co, St; Louis, Mo.) is added to aconcentration of 1 mM and incubation at 37° C. is continued for anadditional 4 hours. The bacterial cells are then pelleted bycentrifugation at 4,000×g for 20 minutes. Extraction of the protein isperformed as described in protocol 10 (“Purification of periplasmicproteins”) in the QIAexpressionist manual (Version 3/99; Qiagen, Inc,Valencia, Calif.). In particular, the cell pellet are resuspended in 30mM Tris-Cl, 20% sucrose, pH 8.0 at 80 ml per gram wet weight of pellet.EDTA is then added to reach a concentration of 1 mM and the cells aregently stirred for 10 minutes. The cell suspension is then centrifugedat 8000×g for 20 minutes at 4° C., and the pellet is resuspended in thesame volume of cold 5 mM MgSO₄. Next, the suspension is stirred gentlyfor 10 minutes, then centrifuged at 8000×g for 20 minutes at 4° C. Thesupernatant is then dialyzed 3 times (at least 6 hours each) against 1 Lof lysis buffer comprising 8 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris-Cl pH8.0. It is to be noted that dialysis is performed here and elsewhere inthe application using Spectra/Por 5,000 Da MW cutoff dialysis tubing(Spectrum Medical Industries, Inc., Houston Tex.).

The cleared lysate obtained above is then subjected to Ni-NTApurification using protocol 14 from the QIAexpressionist manual (Version3/99). Briefly, the purification is performed as follows: 1 ml 50%Ni-NTA slurry (Qiagen, Inc.) is added to 4 ml cleared lysate and mixedgently for 60 minutes on a rotary shaker. The mixture is then loadedinto a 5 ml chromatography column (from Qiagen, Inc) and the flowthrough is collected. The column is washed twice with 4 ml “buffer C” [8M urea, 0.1 M NaH2PO4, 0.01 M Tris-Cl pH 6.3]. The column is then elutedwith 4 washes with 0.5 ml “buffer D” [8 M urea, 0.1 M NaH₂PO₄, 0.01 MTris-Cl pH 5.9], followed by 4 similar washes with “buffer E” [8 M urea,0.1 M NaH₂PO₄, 0.01 M Tris-Cl pH 4.5]. Most of the Aap-6His protein isfound in the buffer E washes. These fractions are pooled and dialyzedagainst three changes of phosphate-buffered saline for 18 hours each,using 5,000 Da MW dialysis tubing (Spectrum).

High quality polyclonal antiserum is raised against the purifiedAap-6His protein fusion by subcutaneous injection of rabbits (asdescribed below). The Aap protein containing the Histidine tag ispurified by chromatography through a Nickel-based column provided byQuiagen, Inc., according to manufacturer's instructions.

After elution from the column, the protein is separated on 10% SDS-PAGEand excised from the gel using a razor blade. The identity of theprotein is confirmed by N-terminal sequence analysis using automatedEdman degradation in the Protein and Nucleic Acid Facility, StanfordUniversity School of Medicine. Approximately 1 mg of the proteincontained within the gel slice was then macerated and injectedsubcutaneously into a 1.5 kg New Zealand White rabbit. 2 weeks later asecond duplicate gel slice containing protein is injected in the rabbitin an identical fashion. 3 weeks after the second injection, the rabbitis bled. Blood is permitted to clot at room temperature overnight, andthe erythrocytes are then pelleted at 3,000×g and the serum iscollected. The serum is stored at −20° C. An aliquot of the serum isabsorbed for cross-reacting antibodies by reacting 1 ml of antiserum(diluted to 10 ml final volume with PBS) overnight with E. coli agarosebeads (Sigma Chemical Co, St. Louis, Mo.) according to manufacturer'sinstructions. The resulting absorbed antiserum is found to react onlywith the Aap-6His protein by Western blot analysis, performed asdescribed above.

Function of Aap

Overnight growth of strain 042 in L-broth at 37° C., produces aggregates(“flocs”) of bacteria that settled to the bottom of the culture tube.042aap produces a larger volume of precipitated material (to the nakedeye) after overnight growth. In addition, 042aap(pAap) produced asimilar amount of culture precipitate compared to strain 042.

Expression of AAF/II Fimbriae

Expression of AAF/II fimbriae is observed by scanning electronmicroscopy. Strains to be examined are grown in L-broth overnight withshaking at 37° C. Bacterial cultures are spotted onto silica chips (TedPella, Inc., Redding, Calif.) previously coated with poly-lysine for 5minutes. The poly-lysine stock consisted of 0.1% poly-lysine (SigmaChemical Co, St. Louis, Mo.) in distilled water. 30 μl of the overnightbacterial culture are spotted onto the chip and allowed to incubate atroom temperature for 5 minutes. Water is withdrawn using a Pasteurpipette and then 2% glutaraldehyde (E.M.S., Inc, Fort Washington, Pa.)in CaCo buffer (0.1 M Ca cacodylate, 3 mM CaCl₂, all reagents fromE.M.S., Inc.) are applied for 1 hour and incubated at room temperature.The liquid is withdrawn and 0.1 M CaCo buffer is applied 3 times forfive minutes each wash. The specimen is then post-fixed with 2% osmiumtetroxide (E.M.S., Inc) in CaCo buffer for 1 hour on ice. The specimensare then rinsed twice with distilled water for five minutes each. Thespecimens are then stained with 2% uranyl acetate (Ted Pella, Inc.) for30 minutes at room temperature, rinsed with 50% ethanol, then dehydratedin a series of ethanol baths for 5 minutes each (50%, 70%, 90%, 100%).The 100% ethanol wash step is performed three times for 5 minutes each.The specimens are then inserted into the critical point dryer (modelCPD-030, Bal-Tec AG, Balzers, Switzerland) until dry, followed bysputter coating in a Desk II Cold Sputtercoater for 90 seconds usingplatinum/paladium for 90 seconds according to manufacturer's protocols(Denton Vacuum, Inc, Moorestown, N.J.). The specimens are then examinedby scanning electron microscopy in a Leo 1550 field emission scanningelectron microscope (Leo Electron Microscopy Inc, Thornwood, N.Y.).

Localization of the Aap Protein

To localize Aap in wild type E. coli 042, cell fractionation experimentsof wild type E. coli 042 and 042aap are performed. Cultures of the testbacteria are inoculated into 5 ml L-broth cultures and incubatedovernight at 37° C. with shaking. For the respective fractions, samplesare prepared as follows:

(1) Supernatant: 100 μl of culture is withdrawn and the bacteria arepelleted by centrifugation. 20 μl of the remaining supernatant isapplied to an SDS-PAGE gel.

(2) Periplasm: The pellet from a centrifuged 5 ml culture is resuspendedin 1 ml 30 mM Tris-HCl, 20% sucrose, pH 8.0. On ice, EDTA (0.5 M stocksolution, Sigma Chemical) is added to bring the final concentration to 1mM. After five minutes on ice, the cells are pelleted by centrifugationand then resuspended in 1 ml cold 5 mM MgSO4 (Sigma). The suspension isstirred for 10 minutes. The cells are then pelleted by centrifugationand 20 μl of the supernatant is run on SDS-PAGE as the periplasmicfraction.

(3) Whole cell: After centrifugation, the remaining pellet is aspiratedand the cell pellet resuspended in an equal volume of SDS-PAGE runningbuffer. The suspension is boiled for 10 minutes. 10 μl of thispreparation is run on the gel.

By running SDS-PAGE gels and extrapolating back to the number of cellsin the original culture, it is estimated that at least 50% of Aapproduced by strain 042 is localized to the culture supernatant and freefrom the bacterial cell. Whole cell lysates of culture pellets indicatethat the majority of cell associated Aap is in the 12 kDa unprocessedcytoplasmic form. It is also determined that the Aap protein has anexcellent signal sequence between residues 21 and 22 of SEQ ID NO: 1.

Role of Aap in Intestinal Adherence

Because of the decrease in aggregation of E. coli 042aap(pAap), theability of the aap mutant to adhere to human mucosa in in vitro organcultures (IVOC) is examined. Colonic samples are obtained with fullyinformed parental consent from pediatric patients undergoing colonoscopyfor possible inflammatory bowel disease. All tissue used in theseexperiments are determined normal by pathologic examination prior toexperimentation.

Endoscopy is performed using an Olympus PCF pediatric endoscope. 2-3 mm²biopsy specimens are taken from the transverse colons from 6 pediatricpatients (5 male:1 female; aged 41-174 months, median age 107 months).Tissue is mounted (mucosal surface upwards) on a foam support(polyethylene packing foam taken from discarded packing material) in apetri dish (VWR Scientific, Dorset, England) at 37° C. in a 95% O₂ and5% CO₂ atmosphere. The tissue is partially submerged in mediumcontaining a 1:1 mixture of NCTC-135 medium and DMEM containing 0.5%d-mannose, with 10% (vol/vol) newborn calf serum (Gibco/BRL,Gaithersburg Md.). The medium is changed every two hours to maintain pHand nutrient supply. Bacteria are grown in brain heart infusion broth(Difco, Becton Dickenson, Franklin Lakes, N.J.) for eighteen hours at37° C. without agitation. 25 μl of the overnight bacterial culture isapplied to the tissue and incubated for eight hours. The tissuespecimens are washed three times in fresh NCTC-135/DMEM medium to removeany non-adherent bacteria. Specimens are then fixed in 3%phosphate-buffered saline glutaraldehyde (Sigma Chemical Co, St. LouisMo.) and postfixed in 1% aqueous osmium tetroxide (mfg). Specimens arethen taken through a graduated series of ethanol and critical pointdried in liquid CO₂, using a Polaron E3000 critical point dryingapparatus. Samples are sputter-coated with gold palladium in a PolaronE5100 series II coating system and examined in a JEOL JSM scanningelectron microscope (JEOL, Inc., Peabody, Mass.). Eight hour pediatricintestinal IVOC are compared with uninoculated controls and wild type E.coli O42.

E. coli 042 wild type adhere to the mucosa with thick aggregates ofbacteria and single bacteria. In contrast, the aap mutant (E. coli042aap) adhered in “super-aggregates” with virtually no single bacteriaon the mucosal surface. Thus, the IVOC experiments illustrate that Aaphelps in the dispersal or non-aggregation of EAEC.

Expression Conditions of aap

Aap is expressed maximally during the logarithmic growth phase. E. coli042 is cultured in L broth at 37° C. with shaking and 2 ml samples arewithdrawn from the culture every two hours. The bacterial cells arepelleted by centrifugation at 10,000×g and 50 μl of the supernatants aremixed with an equal volume of SDS-PAGE loading buffer (BioRad, Valencia,Calif.). The samples are then boiled for 10 minutes and run on a 10%SDS-PAGE gel. The gel is then electroblotted to Nylon membrane andsubjected to immunoblot using anti-Aap antiserum. Aap expression isshown to be the highest during mid-logarithmic phase.

The Usher Chaperone Pathway

Aap has several attributes characteristic of bacterial pilin subunits,including small size, a cleavable signal sequence, two cysteine residuescapable of forming a disulfide loop, and C-terminal glycine residueswhich could indicate association with a PapD-like chaperone protein.

It has been shown that AAF/II expression is dependent on the PapD-likechaperone AafD and the PapC usher homolog AafC. (Elias, et al., JBacteriol 181:1779-85 (1999)). Organization of biogenesis genes forAggregative Adherence Fimbria II defines a virulence gene cluster inenteroaggregative E. coli. (Id.).

Mutants of strain 042 with insertions of pJP5603 are examined for theirabilities to secrete Aap into the intracellular space. (Id.). The methodfor the insertion of pJP5603 is described above.

Aap was detected in supernatants of E. coli 042 and E. coli 042 withinsertional mutations in the aafD, aafC, and aafA genes. Aap isundetectable in the aafD and aafC mutants. However, Aap is secreted atgreater than wild type levels in aafA mutants (i.e., deficient inexpression of the AAF/II major fimbrial subunit).

To further demonstrate this point, the following experiments areconducted to determine the presence of the Aap protein in cultures of E.coli HS carrying plasmid pAA2 and pAA2 with mutations in aafC, aafD andaafA. Identical results are observed when compared with the wild typestrain, indicating that Aap expression is dependent on theusher-chaperone system of E. coli strain 042, but not on the expressionof the AAF/II fimbriae themselves.

Diagnosis of EAEC Infection

Although the diagnosis of an EAEC infection is described below using anantibody specific for Aap, antibodies to AatP, AatA, AatB, AatC, andAatD can be used for diagnosis in a similar manner.

Individuals with EAEC will most likely pass in their stools EAEC whichexpress the Aap protein on the bacterial surface. Therefore, detectionof E. coli expressing Aap, by immunologic or genetic methods willdiagnose EAEC infection. Immunologic detection can be performed by theslide agglutination method, using antibodies to Aap. The antibodies canbe used alone or conjugated to latex beads.

Because the aap gene is present only in EAEC strains, the presence of E.coli carrying the aap gene will diagnose an EAEC infection. One usefulway to find the aap gene in an E. coli colony is via polymerase chainreaction, PCR. PCR could be performed on E. coli isolated from thestools of patients with diarrhea. A positive reaction would diagnose theinfection. A patient's stool would be plated on MacConkey medium andincubated overnight at 37° C. After such incubation, the plate would beexamined and lactose positive colonies would be picked with a toothpickand resuspended in 500 mcl of distilled water, then boiled for 10 min. 2mcl of this preparation would then be added to an aqueous buffercontaining 10 mM Tris-HCl [pH 8.3], 50 mM KCl, 2 mM MgCl₂, 100 μM eachdATP, dCTP, dGTP, and dTTP, 2.5 U of Ampli Taq polymerase [Perkin-Elmer,Norwalk, Conn.]), 25 pmol of each aap primer. The sequences of PCRprimers are 5′-TTGTTATCTTTTCTGGCATCTTGGGT-3′; (SEQ ID NO: 18) and5′-GAGGGGGTAACAACCCCTTTGAAGT-3′. (SEQ ID NO: 19)The reaction mixture is heated to 94° C. for 5 min and then subjected to35 cycles (94° C. for 30 s, 50° C. for 1 min, and 72° C. for 45 s) ofamplification, and then to a final extension at 72° C. for 7 min in aDNA thermal cycler. Then 10 μl of each of the amplified PCR products isadded to a 1% agarose electrophoresis gel with a 1-kb DNA molecularweight marker (Gibco BRL). A positive reaction is defined as thepresence of the PCR product of the expected size of 0.4 kb.

Colony blot hybridization is performed by standard methods (Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, 2001; pp. 1.126-1.142). A piece of nitrocellulosepaper (Millipore HAWP) is layered onto an L-agar plate. Bacterialcolonies to be tested are inoculated onto the nitrocellulose usingtoothpicks. The inoculated plate is incubated overnight at 37° C. Afterovernight incubation, the nitrocellulose paper is saturated with 10% SDSin water for 3 min, then saturated with denaturing solution [0.4 MNaOH/10 mM EDTA] for 5 min. After this step, the filters are wetted withneutralizing solution [1M Tris pH 7.0, 1.5M NaCl] two times for fiveminutes each. After these treatments, the filter is wetted with 2×SSPE[0.3 M NaCl, 20 mM NaH₂PO₄, 2 mM EDTA, pH 7.0] for 5 min. The filter isthen dried before being baked at 80° C. under vacuum.

For the hybridization reaction, the filters are first wetted for 5 minin 2×SSC [0.3 M NaCl/0.03 M trisodium citrate], then are incubated at50° C. for 60 min in 3×SSC [0.45 M NaCl, 0.045 M trisodium citrate]/1%SDS, with 4× Denhardt solution added [where 100× Denhardt solutioncomprises 2% Ficoll 400, 2% polyvinylpyrrolidone, and 20 g/L Bovineserum albumin]. 100 ug/ml denatured salmon sperm DNA is added. Forhybridization, 25 ng of probe fragment (the PCR product described abovein this section are purified by agarose gel electrophoresis using lowmelting temperature agarose, and extraction from the gel by dissolvingthe gel slice at 70° C. for one hr; the DNA is then labeled by randompriming with ³²P d-CTP by using a commercially available labeling kit(Pharmacia Biotech, Piscataway, N.J.). Unincorporated nucleotides areremoved by passage through Sephadex G50 micro-columns (PharmaciaBiotech). The radiolabeled probe fragment is then added directly to theblot already in hybridization mix above. The blot is left to hybridizeat 65° C. overnight, after which time, the blot is washed three timeswith 0.1×SSC/0.1% SDS at 65° C., and exposed to x-ray film at −80° C.overnight. Strains that had been characterized in previous studies wereused as controls. A darkening of the film in the area of an E. colicolony is interpreted as a positive test, thereby diagnosing an EAECinfection in the patient from whom the E. coli was isolated.

Immunogenic Composition

Although an immunogenic composition is described below with respect toAap, an immunogenic composition using any of the other proteinsdescribed herein is made in a like manner.

An immunogenic composition can be made using Aap by administering thepurified protein by injection along with an adjuvant. One such adjuvantused in animals and humans is aluminum hydroxide (alum). One milligramof aluminum hydroxide is mixed with 50 mcg of protein antigen in 1 ml of0.9% saline and the mixture vortexed then incubated at room temperaturefor 20 min. The mixture is centrifuged at 10,000×g for 10 min. Thesupernatant is withdrawn and saved at 4° C. To test immunogenicity, themixture can be injected subcutaneously into rabbits in two 0.5 mlinjections. For human injection, the preparation would be made inexactly the same way, but under the conditions of Good ManufacturingPractices. The antigen could be injected intramuscularly to humans as asubunit vaccine.

Nucleotide Immunogenic Compositions

Although an immunogenic composition is described below with respect toaap, an immunogenic composition using any of the other genes describedherein is made in a like manner.

The aap gene could be delivered as a DNA vaccine to induce antibodies tothe Aap protein. The aap gene would be amplified using the primers (SEQID NO: 20) 5′-ACAAGCTTGCAAAAAATTAAGTTTGTTATC-3′ and (SEQ ID NO: 17)5′-CGGGATCCAACCCATTCGGTTAGAGC-3′;use of these primers results in the incorporation of HindIII and BamHIsites onto the DNA fragment. The PCR product would be digested withthese two enzymes by manufacturer's protocols (New England Biolabs) andthe fragment would be ligated with vector pcDNA2, previously digestedwith the same enzymes. The reaction would take place using DNA ligasealso from New England Biolabs using manufacturer's suggested protocols.This experiment results in thus placing the aap gene downstream of theCMV promoter of this vector. The ligation mix would be transformed intoE. coli DH5α, selecting for ampicillin resistance. The plasmid couldthen be purified by standard alkaline lysis techniques, and would beready for injection as a DNA vaccine. The DNA would be dissolved inendotoxin-free PBS at a concentration of 1 mg/ml. 50 μl of thissuspension could be injected into mouse thigh muscle to testimmunogenicity. Approximately 1 ml would serve as a human dose.

EAEC Vaccine

The Aap protein could also serve as an effective vaccine for EAECinfection. It has been shown that the Aap protein is secreted outside ofthe bacterial cell and that inhibition of the function of the protein(by mutagenesis of the gene) changes the ability of the bacterium topenetrate a mucous layer and also changes the morphology of interactionwith the intestinal mucosa. For this reason, it is inferred that Aap isa virulence factor and that the presence of antibodies against theprotein will interfere with the pathogenic process. Thus, expression ofthe Aap protein could serve as a useful component of an EAEC vaccine.

The Aap protein could be expressed as part of a vaccine by engineeringexpression of the protein in a vaccine delivery vector. Most gramnegative vaccine vectors could probably be successfully engineered toaccomplish this. One example of this technology would be to introducethe pAap plasmid into vector vaccine strain CVD 1204 (U.S. Pat. No.5,783,196, issued to Noreiga et al. on Jul. 21, 1998). CVD 1204 is anattenuated Shigella vector vaccine which colonizes the colonic mucosaand induces an antibody response to both Shigella surface antigens andforeign proteins expressed on the bacterial cell surface, includingenterotoxigenic E. coli fimbrial proteins (Altboum et al., Infect Immun69:3150-8 (2001)). The pAap plasmid would be introduced byelectroporation using the method described above in the section“Isolation and Purification of Aap protein and generation ofantibodies”. The vaccine could then be administered orally as describedin the literature (Kotloff et al., Infect Immun 68:1034-9 (2000)) and anantibody response could be detected by Western immunoblots using themethods described above.

Another example of this technology would be to PCR amplified the aapgene from strain 042 using upstream and downstream primers incorporatingBamHI (upstream primer) and NheI (downstream primer) sites. After PCR,the fragment would be purified from an agarose gel and digested withBamHI and NheI. Expression vector pSEC10 would be digested with therestriction enzymes BamHI and NheI, generating two fragments. The largerfragment would be isolated from an agarose gel and ligated with the aapPCR fragment previously digested with BamHI and NheI. Digestion andligation protocols are as described in Ausubel et al, Current Protocolsin Molecular Biology. The ligation mix would then be electroporated intocompetent Shigella flexneri vaccine vector strain CVD 1204.Transformants would be selected on kanamycin containing agar and theplasmid verified by digestion with BamHI and NheI.

The aap expression plasmid in CVD 1204 would comprise an aap-expressingattenuated Shigella strain, which would be expected to express Aap onits surface. This would be verified by Western blot using the methodsdescribed above. 10 ml of L-broth would be inoculated with theAap-expressing pSEC10 clone and permitted to grow overnight. After thisperiod, the cells would be precipitated by centrifugation and washed for10 min in 500 mcl 0.1% Triton X-100 detergent to remove surfacelocalized Aap protein. 20 mcl of this detergent wash would be applied toan SDS-PAGE gel, and subjected to electrophoresis. The gel would betransferred to nitrocellulose and subjected to Western immunoblot usinganti-Aap antiserum as above.

The amplified aap would include its signal sequence, which would mediatesecretion into the periplasm. Although Aap appears to have a dedicatedsecretion system (Aat), it has been shown that there is residualtranslocation to the bacterial cell surface even in the absence of Aat.Therefore, the expression of Aap alone may be sufficient to establishexport.

aat Vaccine

The AatA protein could also serve as an effective vaccine for EAECinfection. We have shown that the AatA protein is secreted to the outermembrane of the bacterial cell and appears to be able to do so in theabsence of other genes. Therefore, the AatA protein could be expressedas part of a vaccine by engineering expression of the protein in avaccine delivery vector without additional proteins. aat can beintroduced into an attenuated EAEC, Salmonella, Shigella, Lactobacillus,or other bacteria. The bacteria is then administered to an animal togenerate an immune response to Aat.

The aatA gene would be PCR amplified from strain 042 using upstream anddownstream primers incorporating BamHI (upstream primer) and NheI(downstream primer) sites. After PCR, the fragment would be purifiedfrom an agarose gel and digested with BamHI and NheI. Expression vectorpSEC10 would be digested with the restriction enzymes BamHI and NheI,generating two fragments. The larger fragment would be isolated from anagarose gel and ligated with the aatA PCR fragment previously digestedwith BamHI and NheI. Digestion, ligation andtransformation/electroporation protocols are as described (Sambrook andRussell, Molecular Cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press, 2001). The ligation mix would then be electroporatedinto competent Shigella flexneri vaccine vector strain CVD 1204.Transformants would be selected on kanamycin containing agar and theplasmid verified by digestion with BamHI and NheI.

The aatA expression plasmid in CVD 1204 would comprise anaatA-expressing attenuated Shigella strain, which would be expected toexpress AatA on its surface (inserted in the outer membrane). This wouldbe verified by Western blot using standard methods (Harlowe and Lane,Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press,1988). 10 ml of L-broth would be inoculated with the AatA-expressingpSEC10 clone and permitted to grow overnight. After this period, thecells would be precipitated by centrifugation and Triton X-100 added tothe supernatant to a final concentration of 2%. The preparation iscentrifuged at 20,000×g for 15 minutes and the pellet applied to anSDS-PAGE gel, then subjected to electrophoresis. The gel would betransferred to nitrocellulose and subjected to Western immunoblot usinganti-AatA antiserum.

When expressed in an attenuated Shigella strain, such as describedabove, guinea pigs, anesthetized subcutaneously with ketamine HCl (40mg/kg) and xylazine (5 mg/kg), are inoculated by intranasaladministration twice on days 0 and 14, with ˜2×10⁹ bacteria (0.1 ml of40OD₆₀₀ nm) of the Shigella AatA strain that had been propagated onTSA/Congo red/guanine containing plates and harvested in PBS. Sera wereobtained on days 0, 14 and 30 by anterior vena cava puncture ofanesthetized animals. The immunized animals were tested for bacterialagglutination, immunoblotting, and inhibition of hemagglutination andadherence of Aat expressing strains to Caco-2 cells. The animals wouldbe tested for immune response to AatA by Western blot as describedabove. This group of animals would then be challenged orally with 10⁹cfu of strain 042; a second group of control animals not immunized wouldalso be challenged with 042. Stool samples from all animals would bescreened for shedding of EAEC by colony hybridization for aap asdescribed above. The presence of bacteria containing aap indicated anEAEC infection.

Mechanism of Action and Importance

It has been shown that enteric pathogens must adhere to the epithelialsurface to effect efficient colonization. (Nataro et al., Clin MicrobiolReviews January; 11(1): 142-201 (1998)). However, it may be important tomodulate that adherence to permit improved spread across the epithelialsurface. Knutton, et al., Molecular Microbiology 33: 499-509 (1999),have shown that the bundle-forming pilus (BFP) of EPEC serves bothaggregate formation and detachment roles, which occur sequentially. Inthis way, EPEC can adhere and multiply early in the pathogeneticsequence, establishing a foothold on the epithelium, but can then “shakeloose” individual progeny that are free to establish new foci ofinfection on the mucosa at some distance. This paradigm is intuitivelybeneficial for the bacteria, and therefore may be a property of manyother mucosal pathogens. Indeed, Benitez, et al. have suggested that theHap mucinase of Vibrio cholerae may serve as an enzymatic “detachase”,the mutation of which results in increased density of bacterialcolonization but also attenuation of virulence. (Infection and Immunity69:6549-53 (2001)). Similarly, the highly conserved Aap protein of EAECmay serve as a modulator of adherence and colonization in EAEC.

Aap is secreted from the bacterial cell and is present free from thecell in the extracellular environment. This evidence suggests that Aapworks in a unique fashion. Aap is more hydrophilic than AAF/II, and thusits presence outside the cell mitigates the high surface hydrophobicityconferred by the expression of the AAF fimbriae. Aap may also possessenzymatic activity as well, but in this case, the substrate would needto be of bacterial origin because Aap effects are illustrated only in acell-free in vitro system. An alternative embodiment is that Aap bindsto the bacterial outer membrane or to the AAF fimbriae themselves,thereby modulating the biophysical characteristics of the bacterialcell. However, Aap does not bind to any cellular component. In anotherembodiment, Aap suppresses the expression of the AAF fimbriae at apost-translational level.

Aap plays a role in the penetration of the mucosal mucous blanket,perhaps through a lessening of particle size. Indeed, as demonstrated ina purified mucin gel column assay, Aap mutants penetrated more slowlythan the wild type parent (see above). Thus, moderation of EAECaggregates could serve to promote penetration of the mucus blanket,and/or promote the dissemination of the organism across the mucosalsurface.

The invention of this application has been described above bothgenerically and with regard to specific embodiments. Although theinvention has been set forth in what is believed to be the preferredembodiments, a wide variety of alternatives known to those of skill inthe art can be selected within the generic disclosure. The invention isnot otherwise limited, except for the recitation of the claims set forthbelow.

1. An isolated polynucleotide molecule comprising a polynucleotide having at least 95% homology to nucleotides 1 to 348 of SEQ ID NO:1.
 2. An isolated polynucleotide molecule comprising a polynucleotide encoding a polypeptide having at least 95% homology to amino acids 1 to 116 of SEQ ID NO:2.
 3. An expression vector comprising an isolated polynucleotide molecule according to claim
 1. 4. An expression vector comprising an isolated polynucleotide molecule according to claim
 2. 5. A isolated host cell comprising an expression vector of claim
 3. 6. A isolated host cell comprising an expression vector of claim
 4. 7. A method of preparing a polypeptide encoded by a polynucleotide having at least 95% homology to nucleotides 1 to 348 of SEQ ID NO:1, comprising culturing the host cell of claim 5 under conditions promoting the expression of said polypeptide and recovering said polypeptide from the cell culture.
 8. A method of preparing a polypeptide having at least 95% homology to amino acids 1 to 116 of SEQ ID NO:2, comprising culturing the host cell of claim 6 under conditions promoting the expression of said polypeptide and recovering said polypeptide from the cell culture.
 9. A composition comprising a pharmaceutically acceptable carrier and an expression vector according to claim 3 or
 4. 10. The composition of claim 9, wherein said composition is in the form of a composition suitable for administration by a route selected from the group consisting of oral administration, nasal administration, inhalation, ophthalmic administration, rectal administration and vaginal administration.
 11. A method of generating an antibody response in an animal comprising administering a composition according to claim 9 to said animal.
 12. A composition comprising a pharmaceutically acceptable carrier and an isolated host cell according to claim 5 or
 6. 13. The composition of claim 12, wherein said composition is in the form of a composition suitable for administration by a route selected from the group consisting of oral administration, nasal administration, inhalation, ophthalmic administration, rectal administration and vaginal administration.
 14. A method of generating an antibody response in an animal comprising administering a composition according to claim 12 to said animal.
 15. A composition comprising a pharmaceutically acceptable carrier and an isolated host cell, wherein said host cell comprises an expression vector comprising: (i) an isolated polynucleotide molecule comprising nucleotides 1-348 of SEQ ID NO:1; or (ii) an isolated polynucleotide molecule encoding amino acids 1 to 116 of SEQ ID NO:2.
 16. The composition of claim 15, wherein said composition is in the form of a composition suitable for administration by a route selected from the group consisting of oral administration, nasal administration, inhalation, ophthalmic administration, rectal administration and vaginal administration.
 17. A method of generating an antibody response in an animal comprising administering a composition according to claim 15 to said animal. 